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Methods in
Molecular Biology 2346
Kursad Turksen Editor
Stem Cell
Renewal
and Cell-Cell
Communication
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in Pub Med.
Stem Cell Renewal and Cell-Cell
Communication
Methods and Protocols
Second Edition
Edited by
Kursad Turksen
Ottawa, ON, Canada
Editor
Kursad Turksen
Ottawa, ON, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1569-0 ISBN 978-1-0716-1570-6 (eBook)
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Preface
Great strides have been made in the field of cell–cell communications with respect to the
identification and characterization of key components of the communication apparatus,
assembly and maintenance of the communications structures, and concomitantly their
roles in not only tissue formation and maintenance but also regeneration and repair. In
this second edition of the Stem Cell Renewal and Cell-Cell Communication volume, I have
brought together a new set of protocols to arm cell biologists with protocols that are
currently being used in a number of well-established laboratories around the world. I
hope that both people already in the field as well as newcomers will benefit from this
compilation, and that the volume will drive continued growth in our understanding of the
crucial biological and physiological roles of cell–cell communications in tissue function and
organismal integrity.
Once again, the protocols gathered here are faithful to the mission statement of the
Methods in Molecular Biology series: They are well-established and described in an easy to
follow, step-by-step fashion so as to be valuable for not only experts but also novices in the
field. That goal is achieved because of the generosity of the contributors who have carefully
described their protocols in this volume, and I am very grateful for their efforts.
My thanks as well go to Dr. John Walker, the Editor-in-Chief of the Methods in
Molecular Biology series, for giving me the opportunity to create this volume and for
supporting me along the way.
I am also grateful to Patrick Marton, the Executive Editor of Methods in Molecular
Biology and the Springer Protocols collection, for his continuous support from idea to
completion of this volume.
A special thank you goes to Anna Rakovsky, Assistant Editor for Methods in Molecular
Biology, for her continuous support from beginning to end of this project.
I would also like to thank David C. Casey, Senior Editor of Methods in Molecular Biology,
for his outstanding editorial work during the production of this volume.
Finally, I would like to thank Anand Ventakachalam and the rest of the production crew
for their work in putting together an outstanding volume.
Ottawa, ON, Canada Kursad Turksen
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Inference of Ligand–Receptor Pairs from Single-Cell Transcriptomics Data. . . . . . . . . 1

Mirjana Efremova and Roser Vento-Tormo
Multiple Imaging Modalities for Cell-Cell Communication via Calcium
Mobilizations in Corneal Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Yoonjoo K. Lee, Kristen L. Segars, and Vickery Trinkaus-Randall
Interactions of Hematopoietic Stem Cells with Bone Marrow Niche. . . . . . . . . . . . . . . 21
Xinghui Zhao, Cuiping Zhang, Xiaojing Cui, and Ying Liang
Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver . . . . . . . . . 35
Amina Mohammadalipour, Miguel F. Diaz, Sumedha Pareek,
and Pamela L. Wenzel
Analysis of Epithelial Architecture and Planar Spindle Orientation
in the Drosophila Wing Disc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Yu-ichiro Nakajima
A Co-culture Model to Study the Effect of Kidney Fibroblast-p90RSK
on Epithelial Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Ling Lin, Samantha White, and Kebin Hu
Calcium Fluorescence Recordings from Neuroepithelial Stem Cells. . . . . . . . . . . . . . . . 73
Masayuki Yamashita
Ultrastructural Analysis of Cell–Cell Interactions in Drosophila Ovary . . . . . . . . . . . . . 79
Matthew Antel, Valentina Baena, Mark Terasaki, and Mayu Inaba
TIRF Microscopy as a Tool to Determine Exosome Composition . . . . . . . . . . . . . . . . . 91
Noa B. Martı́n-Co freces, Daniel Torralba, Marta Lozano-Prieto,
Nieves Fernández-Gallego, and Francisco Sánchez-Madrid
Rapid Visualization of Intracellular Vesicle Events During Synaptic
Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Noa B. Martı́n-Co freces, Amelia Rojas-Gomez, Sara G. Dosil,
Irene Fernandez-Delgado, and Francisco Sánchez-Madrid
Monitoring of Active Notch Signaling in Mouse Bladder Urothelium . . . . . . . . . . . . . 121
Panagiotis Karakaidos and Theodoros Rampias
Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing . . . . . . 135
Gareth W. Price, Joe A. Potter, Bethany M. Williams, Chelsy L. Cliff,
Mark J. Wall, Claire E. Hills, and Paul E. Squires
Isolation and Assessment of Pancreatic Islets Versus Dispersed Beta Cells:
A Straightforward Approach to Examine Cell–Cell Communication . . . . . . . . . . . . . . . 151
Rachel T. Scarl, William J. Koch, Kathryn L. Corbin, and Craig S. Nunemaker
vii
viii Contents
Promoter Pull-Down Assay: A Biochemical Screen for DNA-Binding

Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Ryan R. Chaparian and Julia C. van Kessel
Purification of the Vibrio Quorum-Sensing Transcription Factors LuxR,
HapR, and SmcR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Jane D. Newman and Julia C. van Kessel
Preserving Cytonemes for Immunocytochemistry of Cultured Adherent
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Sally Rogers and Steffen Scholpp
Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate
Registration of Target Single Cells Based on a Triangle Standard Chip
for the Image Analysis of Gap Junctional Communication. . . . . . . . . . . . . . . . . . . . . . . . 191
Mikako Saito
Chemical and Voltage Gating of Gap Junction Channels Expressed
in Xenopus Oocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Camillo Peracchia
The Analysis of Gap Junctional Intercellular Communication Among
Osteocytes in Chick Calvariae by Fluorescence Recovery After Photobleaching . . . . . 215
Ziyi Wang, Yoshihito Ishihara, and Hiroshi Kamioka
Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular
Communication Between Cytotoxic T Cells and Target Tumor Cells . . . . . . . . . . . . . . 225
Mariela Navarrete, Flavio Salazar-Onfray, and Andrés Tittarelli
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Contributors
MATTHEW ANTEL • Department of Cell Biology, University of Connecticut Health Center,

Farmington, CT, USA
VALENTINA BAENA • Department of Cell Biology, University of Connecticut Health Center,
Farmington, CT, USA
RYAN R. CHAPARIAN • Biology Department, Indiana University, Bloomington, IN, USA
CHELSY L. CLIFF • Joseph Banks Laboratories, School of Life Sciences, University of Lincoln,
Lincoln, UK
KATHRYN L. CORBIN • Department of Biomedical Sciences Heritage College of Osteopathic
Medicine, Ohio University, Athens, OH, USA; Diabetes Institute, Heritage College of
Osteopathic Medicine, Ohio University, Athens, OH, USA
XIAOJING CUI • Department of Toxicology and Cancer Biology, University of Kentucky,
Lexington, KY, USA
MIGUEL F. DIAZ • Department of Integrative Biology and Pharmacology, McGovern Medical
School, University of Texas Health Science Center at Houston, Houston, TX, USA; Center
for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular
Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA
SARA G. DOSIL • Servicio de Inmunologı́a, Hospital Universitario de la Princesa,
Universidad Autonoma de Madrid, Instituto Investigacion Sanitaria Princesa (IIS-IP),
Madrid, Spain; Vascular Pathophysiology Area, Centro Nacional Investigaciones
Cardiovasculares (CNIC), Madrid, Spain
MIRJANA EFREMOVA • Barts Cancer Institute, Queen Mary University of London, London,
UK; Wellcome Sanger Institute, Cambridgeshire, UK
IRENE FERNANDEZ-DELGADO • Servicio de Inmunologı́a, Hospital Universitario de la
Princesa, Universidad Autonoma de Madrid, Instituto Investigacion Sanitaria Princesa
(IIS-IP), Madrid, Spain; Vascular Pathophysiology Area, Centro Nacional Investigaciones
NIEVES FERNÁNDEZ-GALLEGO • Servicio de Inmunologı́a, Hospital Universitario de la
CLAIRE E. HILLS • Joseph Banks Laboratories, School of Life Sciences, University of Lincoln,
Lincoln, UK
KEBIN HU • Department of Medicine, The Pennsylvania State University College of
Medicine, Hershey, PA, USA; Department of Cellular and Molecular Physiology, The
Pennsylvania State University College of Medicine, Hershey, PA, USA
MAYU INABA • Department of Cell Biology, University of Connecticut Health Center,
Farmington, CT, USA
YOSHIHITO ISHIHARA • Department of Orthodontics, Okayama University Graduate School of
Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
HIROSHI KAMIOKA • Department of Orthodontics, Okayama University Graduate School of
Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
PANAGIOTIS KARAKAIDOS • Biomedical Research Foundation of the Academy of Athens,
Athens, Greece
ix
x Contributors
WILLIAM J. KOCH • Department of Biomedical Sciences Heritage College of Osteopathic

Medicine, Ohio University, Athens, OH, USA; Translational Biomedical Sciences,
Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA; Diabetes
Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA
YOONJOO K. LEE • Departments of Pharmacology and Biochemistry, Boston University School
of Medicine, Boston, MA, USA
YING LIANG • Department of Toxicology and Cancer Biology, University of Kentucky,
Lexington, KY, USA
LING LIN • Department of Medicine, The Pennsylvania State University College of Medicine,
Hershey, PA, USA; Department of Cellular and Molecular Physiology, The Pennsylvania
State University College of Medicine, Hershey, PA, USA
MARTA LOZANO-PRIETO • Servicio de Inmunologı́a, Hospital Universitario de la Princesa,
NOA B. MARTÍN-CÓFRECES • Servicio de Inmunologı́a, Hospital Universitario de la Princesa,
Cardiovasculares (CNIC), Madrid, Spain; Centro de Investigacion Biomédica en Red
Cardiovascular (CIBERCV), Madrid, Spain
AMINA MOHAMMADALIPOUR • Department of Integrative Biology and Pharmacology,
McGovern Medical School, University of Texas Health Science Center at Houston, Houston,
TX, USA
YU-ICHIRO NAKAJIMA • Frontier Research Institute for Interdisciplinary Sciences, Tohoku
University, Sendai, Japan; Graduate School of Life Sciences, Tohoku University, Sendai,
Japan
MARIELA NAVARRETE • Millennium Institute on Immunology and Immunotherapy, Faculty
of Medicine, Universidad de Chile, Santiago, Chile; Disciplinary Program of Immunology,
Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, Santiago,
Chile
JANE D. NEWMAN • Biology Department, Indiana University, Bloomington, IN, USA
CRAIG S. NUNEMAKER • Department of Biomedical Sciences Heritage College of Osteopathic
Medicine, Ohio University, Athens, OH, USA; Diabetes Institute, Heritage College of
Osteopathic Medicine, Ohio University, Athens, OH, USA
SUMEDHA PAREEK • Immunology Program, MD Anderson Cancer Center UTHealth
Graduate School of Biomedical Sciences, Houston, TX, USA
CAMILLO PERACCHIA • Department of Pharmacology and Physiology, School of Medicine and
Dentistry, University of Rochester, Rochester, NY, USA
JOE A. POTTER • Joseph Banks Laboratories, School of Life Sciences, University of Lincoln,
Lincoln, UK
GARETH W. PRICE • Joseph Banks Laboratories, School of Life Sciences, University of Lincoln,
Lincoln, UK
THEODOROS RAMPIAS • Biomedical Research Foundation of the Academy of Athens, Athens,
Greece
SALLY ROGERS • Living Systems Institute, School of Biosciences, College of Life and
Environmental Sciences, University of Exeter, Exeter, UK
AMELIA ROJAS-GOMEZ • Servicio de Inmunologı́a, Hospital Universitario de la Princesa,
Contributors xi

MIKAKO SAITO • Department of Biotechnology and Life Science, Tokyo University of
Agriculture and Technology, Tokyo, Japan
FLAVIO SALAZAR-ONFRAY • Millennium Institute on Immunology and Immunotherapy,
Faculty of Medicine, Universidad de Chile, Santiago, Chile; Disciplinary Program of
Immunology, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile,
Santiago, Chile
FRANCISCO SÁNCHEZ-MADRID • Servicio de Inmunologı́a, Hospital Universitario de la
Cardiovasculares (CNIC), Madrid, Spain; Centro de Investigacion Biomédica en Red
Cardiovascular (CIBERCV), Madrid, Spain
RACHEL T. SCARL • Department of Biomedical Sciences Heritage College of Osteopathic
Medicine, Ohio University, Athens, OH, USA
STEFFEN SCHOLPP • Living Systems Institute, School of Biosciences, College of Life and
Environmental Sciences, University of Exeter, Exeter, UK
KRISTEN L. SEGARS • Departments of Pharmacology and Biochemistry, Boston University
School of Medicine, Boston, MA, USA
PAUL E. SQUIRES • Joseph Banks Laboratories, School of Life Sciences, University of Lincoln,
Lincoln, UK
MARK TERASAKI • Department of Cell Biology, University of Connecticut Health Center,
Farmington, CT, USA
ANDRÉS TITTARELLI • Programa Institucional de Fomento a la Investigacion, Desarrollo
e Innovacion (PIDi), Universidad Tecnologica Metropolitana (UTEM), Santiago, Chile
DANIEL TORRALBA • Servicio de Inmunologı́a, Hospital Universitario de la Princesa,
VICKERY TRINKAUS-RANDALL • Departments of Pharmacology and Biochemistry, Boston
University School of Medicine, Boston, MA, USA
JULIA C. VAN KESSEL • Biology Department, Indiana University, Bloomington, IN, USA
ROSER VENTO-TORMO • Wellcome Sanger Institute, Cambridgeshire, UK
MARK J. WALL • School of Biomedical Science, University of Warwick, Coventry, UK
ZIYI WANG • Department of Orthodontics, Okayama University Graduate School of
Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; Research Fellow of
Japan Society for the Promotion of Science, Tokyo, Japan
PAMELA L. WENZEL • Department of Integrative Biology and Pharmacology, McGovern
Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA;
Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of
Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX,
USA; Immunology Program, MD Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences, Houston, TX, USA
SAMANTHA WHITE • Department of Medicine, The Pennsylvania State University College of
Medicine, Hershey, PA, USA; Department of Cellular and Molecular Physiology, The
Pennsylvania State University College of Medicine, Hershey, PA, USA
BETHANY M. WILLIAMS • Joseph Banks Laboratories, School of Life Sciences, University of
Lincoln, Lincoln, UK
xii Contributors
MASAYUKI YAMASHITA • Center for Basic Medical Research, International University of

Health and Welfare, Ohtawara, Japan
CUIPING ZHANG • Department of Toxicology and Cancer Biology, University of Kentucky,
Lexington, KY, USA
XINGHUI ZHAO • Department of Toxicology and Cancer Biology, University of Kentucky,
Lexington, KY, USA
Methods in Molecular Biology (2021) 2346: 1–10
DOI 10.1007/7651_2020_343
© Springer Science+Business Media, LLC 2021
Published online: 25 February 2021
Inference of Ligand–Receptor Pairs from Single-Cell

Transcriptomics Data
Mirjana Efremova and Roser Vento-Tormo
Abstract
Cell–cell communication is crucial for development and tissue homeostasis in multicellular organisms.
Single-cell transcriptomics has emerged as a revolutionary technique for dissecting cellular compositions
and potential cell–cell communication events via ligand–receptor pairs. To provide a systematic characteri-
zation of intercellular communication, we developed a framework to map cell–cell communication events
mediated by ligand–receptor interactions across different cell types using single-cell transcriptomics data.
Our repository of ligands, receptors and their interactions is integrated with a computational approach to
identify cell-type specific and biologically relevant interactions. Here, we summarize the structure and
content of our repository and present a practical guide for inferring cell–cell communication networks from
single-cell RNA sequencing data.
Key words Cell–cell communication, Single-cell RNA sequencing, Receptors, Ligands
1 Introduction
Intercellular communication underlies numerous biological pro-

cesses including coordination of cellular fate, behavior, and
response to neighboring cells, in healthy and diseased conditions.
Single-cell transcriptomics, by measuring the expression of ligands
and receptors in individual cell types, has provided powerful means
for mapping interactions and inferring cellular communication net-
works [1, 2].
To provide a systematic characterization of cell–cell communi-
cation, we developed CellPhoneDB, a public repository of ligands,
receptors and their interactions, integrated with a statistical frame-
work to identify interactions enriched on specific cell types
[2, 3]. Our database relies on public resources as well as manual
curation and takes into account the subunit architecture of ligands
and receptors, to accurately represent heteromeric complexes (see
Notes 1 and 2).
To infer intercellular communication networks between differ-
ent cell types, we consider the expression levels of ligands and
receptors within each cell type, and use empirical shuffling to
1
2 Mirjana Efremova and Roser Vento-Tormo
calculate which ligand–receptor pairs display significant cell-type

specificity. Briefly, we randomly permute the cluster labels of all
cells and calculate the mean of the average ligand expression level in
a cell type and the average receptor expression level in the interact-
ing cell type, generating a null distribution. By calculating the
proportion of the means which are as or higher than the actual
mean, we obtain a p-value for the likelihood of cell-type specificity
of each ligand–receptor complex. In this way, our method predicts
biologically relevant molecular interactions that can be visualized
using intuitive tables and plots.
Our tool is available as a user-friendly interface at cellphonedb.
org and as a python package on github (https://github.com/
Teichlab/cellphonedb).
2 Materials
CellPhoneDB can be used either through the interactive website

(https://www.cellphonedb.org/) or as a Python package using the
user’s computer/cloud/farm. The Python package is recom-
mended for large datasets (datasets larger than 10 GB).
2.1 Input Data Files – Metadata input file: cell type annotation file containing cluster
annotation of each cell. Clustering can be performed with stan-
dard packages for scRNA-seq analysis such as Seurat [4],
SCANPY [5]. The metadata file has two columns: “Cell”,
which represents the name of the cell, and “cell_type”, which
represents the name of the cluster. Accepted formats are .csv (for
comma separated); .txt, .tsv, .tab (for tab separated); and pickle.
– Counts input file: single-cell RNA sequencing (scRNA-seq)
count data with gene expression values where cells are in col-
umns are cells and genes are in rows. Accepted gene names
identifiers include Ensembl IDs, gene names and hgnc_symbol
annotations. Use of normalized count data is recommended.
Accepted formats are .csv (for comma separated); .txt, .tsv, .tab
(for tab separated); and pickle (see Notes 3 and 4).
2.2 Software – Python 3.5 or higher.

– SQLAlchemy (see Note 5).
– SQLite.
2.3 Hardware – Linux or MAC OS.

Inference of Ligand–Receptor Pairs from Single-Cell Transcriptomics Data 3
3 Methods
3.1 Installation We recommend using a virtual environment, but this is optional

(step 1).
1. Create and activate a python virtual environment.
python -m venv cpdb-venv
source cpdb-venv/bin/activate
2. Install CellPhoneDB.
pip install cellphonedb
3.2 Running in the Run CellPhoneDB with statistical analysis using the input file
Statistical Analysis names (specifying the full path to the files).
Mode
cellphonedb method statistical_analysis usermetafile.txt
usercountsfile.txt
Optional parameters:
--project-name: Project’s name. A subfolder with this identity

is created in the output folder. ./out is used by default.
--iterations: Number of iterations for statistical analysis.
1000 interactions are used by default.
--threshold: % of cells expressing the specific ligand or
receptor
--result-precision: Number of decimal digits in results.
3 decimal digits are used by default.
--counts-data: [ensembl | gene_name | hgnc_symbol] Gene iden-
tifiers used in the counts data
--output-path: Directory where the results will be allocated
(the directory must exist) ./out is used by default.
--output-format: Output format of the results files (extension
will be added to filename if not present). txt is used by
default.
--means-result-name: Name of the means result file. means.txt
is used by default.
--significant-mean-result-name: Name of the significant means
result file. Significant_means.txt is used by default
--deconvoluted-result-name: Name of the deconvoluted result
file. deconvoluted.txt is used by default
--verbose/--quiet: Print or hide cellphonedb logs [verbose]
--pvalues-result-name: Name of the pvalues result file. pva-
lues.txt is used by default
--debug-seed: Debug random seed -1. To disable it please use a
value >=0 . -1 is used by default
--threads: Number of threads to use. >=1. 4 is used by default.
l A usage example to set output path, number of iterations, and

number of threads:
mkdir custom_folder
cellphonedb method statistical_analysis
usermetafile.txt usercountsfile.txt --output-
path=custom_folder --iterations=10 --threads=2
3.3 Subsampling and To improve the speed and efficiency and reduce memory require-
Statistical Analysis ments when analyzing large datasets, we implemented a subsam-
pling algorithm [6] in our framework. When using the subsampling
option, the user needs to specify whether the input count data was
log-transformed.
Run CellPhoneDB with statistical analysis using the input files
and add --subsampling with subsampling-specific parameters.
usermetafile.txt usercountsfile.txt --
subsampling --subsampling-log true
In addition to the parameters described in step 3, there are also

subsampling specific parameters.
--subsampling-log: Enable log transformation for
non-log transformed data inputs. This is a mandatory
parameter.
--subsampling-num-pc: Subsampling NumPC argument
--subsampling-num-cells: Number of cells to
subsample to. 1/3 of the cells is used by default.
3.4 Normal Mode, Run CellPhoneDB without statistical analysis using the input files
Without Statistical and a defined --threshold parameter. The parameters are the same
Analysis as described in step 3, with the exception of --pvalues-result-name,
--threads, and --debug-seed which are specific for the statistical
analysis and should not be used here.
cellphonedb method analysis usermetafile.txt
usercountsfile.txt
3.5 Visualization The user can visualize the analysis results using dot plots and
heatmaps.
1. For dot plot visualization, run the dot plot visualization
command using the means.csv and pvalues.scv output files.
This visualization is only relevant for the statistical analysis
mode.
cellphonedb plot dot_plot
Dot plot specific parameters:

--means-path: The means output file. ./out/means.txt
is used by default.
--pvalues-path: The pvalues output file
./out/pvalues.txt is used by default.
--output-path: Output folder. ./out is used by default.
--output-name: Name of the output plot. plot.pdf is
used by default. Available output formats are
restricted to those supported by R’s ggplot2 package
(e.g. pdf, png, jpeg)
--rows: File with a list of rows to plot, one per line
--columns: File with a list of columns to plot, one
per line
--verbose / --quiet: Print or hide cellphonedb logs [verbose]
To plot only specific rows/columns, use text files with a list

of interactions/celltype–celltype comparisons (each interac-
tion/comparison in a new row).
cellphonedb plot dot_plot --rows rows.txt --columns
columns.txt
2. For heatmap visualization, run the heatmap visualization com-

mand, using the pvalues.scv output file. This visualization is
only relevant for the statistical analysis mode.
cellphonedb plot heatmap_plot meta_data
Specific parameters for the heatmap plot:
--pvalues-path: The pvalues output file.

./out/pvalues.txt is used by default.
--output-path: Output folder. default: ./out is used by
default.
--count-name: Filename of the output plot
heatmap_count.pdf is used by default.
--log-name: Filename of the output plot using log-
count of interactions. heatmap_log_count.pdf is used by
default
--count-network-name: Filename of the output network
file. network.txt is used by default.
--interaction-count-name: Filename of the output
interactions-count file. interaction_count.txt is used by
default.
--verbose / --quiet: Print or hide cellphonedb logs
[verbose]
3.6 Different For reproducibility of their analysis, the users have access to differ-
Versions of the ent versions of the CellPhoneDB databases and they can specify
CellPhoneDB Database which version they prefer to use. The users can list and download
available versions of the database from a remote repository (the
CellPhoneDB official available data).
1. Use a specific database version by adding the parameter --
database <version_or_file> to the command “cellphonedb
method”.
usermetafile.txt usercountsfile.txt --database=v0.0.4
The –database <version_or_file> parameter can be either a

database file, in which case it will be used as it is, or a version
number, in which case the version that matches the specified
version parameter will be used. If the specified database version
does not exist locally on the user’s computer it will be down-
loaded from the remote repository. If the --database argument
is not specified, the latest available local database version will be
used for the analysis. The downloaded database versions will be
stored in a folder under ~/.cpdb/releases.
2. List available database versions from the remote CellPhoneDB
repository.
cellphonedb database list_remote
3. List available versions from the local repository, stored locally

on the user’s computer.
cellphonedb database list_local
4. Download a specific version from the remote repository.

cellphonedb database download
or
cellphonedb database download --version
<version_spec|latest>
version_spec must be one of the database versions in the

database listed by the list_remote command. The latest avail-
able version will be downloaded by default. This can be done
also by using latest as a version_spec.
3.7 Generating a Users can also create a custom database, using their own input files.
User-Specific They can choose whether to merge their database with the current
Database CellPhoneDB input files or to only use their own database (see
Note 6).
Generate a custom database with user-specific input files.
cellphonedb database generate

Database generate specific parameters:
--user-protein: Protein input file

--user-gene: Gene input file
--user-complex: Complex input file
--user-interactions: Interactions input file
--fetch: Specific lists can be downloaded from
original sources while creating the database, e.g.,
uniprot, ensembl. The input tables included in the
CellPhoneDB package will be used by default. To
enable downloading an updated copy from the remote servers
--fetch must be appended to the command
--result-path: Output folder
--log-file: Log file
The database file will be generated in the --result-path folder

(by default in the folder “out” if an output folder is not specified)
with cellphonedb_user_{datetime}.db. The user defined input
tables will be by default merged with the current CellPhoneDB
input tables. To use this database, use the --database parameter
when running the “cellphonedb method” command:

in/example_data/test_meta.txt
in/example_data/test_counts.txt --database
out/cellphonedb_user_2019-05-10-11_10.db
l Use only user-specific interactions without merging with the

current CellPhoneDB database (using custom_interaction_file.
csv as a comma separated input file with a list of interactions—
with mandatory columns as described in [3]).
cellphonedb database generate --user-
interactions custom_interaction_file.csv
--user-interactions-only
l Add or modify some existing interactions (using custom_inter-

action_file.csv as a comma separated input file with a list of
interactions to add/modify—with mandatory columns as
described in [3]):
cellphonedb database generate --user-interactions
custom_interaction_file.csv
For duplicated interactions, the user specified interactions

will overwrite the CellPhoneDB interactions.
l Modify any protein data (using custom_protein_file.csv as a
comma separated input file with a list of proteins to over-
write—with mandatory columns as described in [3]).
cellphonedb database generate --user-protein

custom_protein_file.csv
l Update database from remote servers, for example from

UniProt, IMEx, and ensembl.
cellphonedb database generate –fetch
3.8 Help Option Get a detailed description of the mandatory and optional para-
meters using the help option.
cellphonedb method statistical_analysis metafile.txt

countsfile.txt --help
3.9 Interactive Web The web interface includes input forms for the user to specify
Portal analysis parameters before submission. Downstream calculations
are performed on the application’s servers.
1. Upload your input files in the “Exploring your scRNAseq” tab.
You can get an update when the analysis is finished by providing
your email address.
2. When the analysis has finished, the “significant_means” results
table will appear. Results can be downloaded and the current
view can be changed by clicking on the ”Data Shown” button.
To display detailed information for the specific interaction pair,
you can click on any field from the id_cp_interaction column.
3. To visualise the results, go to the “Plots” tab and choose the
type of plot you would like to be generated. For plotting dot
plots, select specific columns and rows of the interactions you
are interested in.
The online results viewer allows the user to select specific
columns to be displayed in each table.
4 Notes
1. CellPhoneDB stores ligand–receptor interactions and informa-

tion related to interacting molecules. For example, we anno-
tated the subunit architecture of each of the interacting
partners as well as multiple gene and protein identifiers. All
this information is stored in four main .csv data files that are
uploaded into the database: “gene_input.csv”, “protein_input.
csv”, “ complex_input.csv”, and “interaction_input.csv”.
The “gene_input” file stores information related to the
gene nomenclature. This information is key to establishing a
connection within the scRNA-seq count data and the
interaction pairs stored in the database as proteins. Specifically,

it stores: (a) gene name (“gene_name”), (b) UniProt identifier
(“uniprot”), (c) HUGO nomenclature committee symbol
(HGNC) (“hgnc_symbol”), and (d) gene ensembl identifier
(ENSG) (“ensembl”). The “protein_input” file stores the
identity of proteins and combines manual annotation of the
protein identities (based on bibliography or uniprot descrip-
tion) as well as data downloaded directly from uniprot. The
“complex_input” file stores the identity of each of the multi-
subunit proteins that will form the heteromeric ligands and
receptors. A specific name is given to each heteromeric com-
plex. Finally, the “interaction_input” file contains the pro-
tein–protein interactions stored in CellPhoneDB. For binary
interactions, each interaction partner is annotated by their
UniProt identifier. For interactions involving heteromers, the
name of the complex is introduced. The identity of the com-
plex is defined in “complex_input”. Each interaction has a
unique identifier (“id_cp_interaction”) generated automati-
cally by the pipeline.
For further details on the content of the tables, please refer
to [3].
2. CellPhoneDB is a curated database, which means that only
information supported by bibliography is stored. This informa-
tion is reviewed internally by members of our team and there-
fore, could be biased toward specific expertise (e.g.,
immunology, development). We encourage users sending
interacting proteins involved in cell–cell communication,
together with a DOI of the publication supporting this infor-
mation. Information can be shared by e-mail, the form avail-
able at www.cellphonedb.org or pull request to the
CellPhoneDB data repository (https://github.com/Tei-
chlab/cellphonedb-data). All the information will be checked
by one of our team members and uploaded into the database.
3. CellPhoneDB was created for scRNA-seq but can be used for
other types of data, for example bulk RNA-seq data from pure
cell populations.
4. CellphoneDB was created using human-specific ligand–recep-
tor interactions, but it can also be applied on datasets from
other species by using orthologs.
5. CellPhoneDB data is stored in an SQLite relational database
(https://www.sqlite.org/). The structure and logic of the
database was built using SQLAlchemy (www.sqlalchemy.org)
and Python 3. All the code is open source and available through
https://github.com/Teichlab/cellphonedb. For further
guidance into the internal structure of the database, please
refer to [3].
6. CellPhoneDB users can also generate their own cell–cell inter-

actions files using our github tool (https://github.com/Tei-
chlab/cellphonedb). For the lists to work in our database, the
format of the users’ lists must be compatible with the input files
of our database. Please refer to [3] for further information.
Acknowledgments
We thank Sarah Teichmann for useful guidance on the method and

curation of protein–protein interactions; Gavin J. Wright, Laura
Wood, and Gerard Graham for advice on protein–protein interac-
tions; and Miquel Vento-Tormo and YDEVS members for their
help with the webserver and the implementation of the code in
github. M.E. is funded by a Barts Charity Lectureship (grant
MGU045). The project was supported by Wellcome Sanger core
funding (no. WT206194) and a Wellcome Strategic Support Sci-
ence award (no. 211276/Z/18/Z).
References
1. Camp JG et al (2017) Multilineage communica- 4. Satija R, Farrell JA, Gennert D, Schier AF, Regev
tion regulates human liver bud development A (2015) Spatial reconstruction of single-cell
from pluripotency. Nature 546:533–538 gene expression data. Nat Biotechnol
2. Vento-Tormo R et al (2018) Single-cell recon- 33:495–502
struction of the early maternal-fetal interface in 5. Wolf FA, Angerer P, Theis FJ (2018) SCANPY:
humans. Nature 563:347–353 large-scale single-cell gene expression data anal-
3. Efremova M, Vento-Tormo M, Teichmann SA, ysis. Genome Biol 19:15
Vento-Tormo R (2020) CellPhoneDB: inferring 6. Hie B, Cho H, DeMeo B, Bryson B, Berger B
cell-cell communication from combined expres- (2019) Geometric sketching compactly sum-
sion of multi-subunit ligand-receptor com- marizes the single-cell transcriptomic landscape.
plexes. Nat Protoc 15:1484–1506 Cell Syst 8:483–493
Methods in Molecular Biology (2021) 2346: 11–20
DOI 10.1007/7651_2020_329
© Springer Science+Business Media New York 2020
Published online: 07 November 2020
Multiple Imaging Modalities for Cell-Cell Communication

via Calcium Mobilizations in Corneal Epithelial Cells
Yoonjoo K. Lee, Kristen L. Segars, and Vickery Trinkaus-Randall
Abstract
Chemical indicators are used to study calcium signaling events in the context of live cell imaging. Fluo-3
AM, Fluo-4 AM, and Cal-520 AM are three commonly used fluorescent indicators derived from the
calcium chelator BAPTA. Here we describe sample protocols that detail how these indicators are used in
in vitro and ex vivo experiments to analyze the role of calcium mobilizations in cell-cell communication and
coordinated cellular motility in the context of wound healing.
Key words Ex vivo in situ analysis, Confocal imaging, Calcium mobilization, Cell-cell communica-
tion, Live cell imaging, Image analysis
1 Introduction
What do neurotransmitter release, skeletal muscle contraction, and

signal transduction of Gq-type G protein-coupled receptors
(GPCRs) have in common? These processes all take advantage of
changes in the intracellular concentration of calcium ions to cause
changes in the cell. In the cytosol, the concentration of calcium ions
is tightly maintained at 100 nM, roughly 20,000–100,000-fold
lower than the concentration in the extracellular space [1]. Due
to the role of calcium ions in regulating enzyme and protein activ-
ity, small alterations in this concentration can cause large changes in
gene transcription, protein activity, and cellular function. In mus-
cles, calcium released from the sarcoplasmic reticulum binds to
troponin [1–3]. This causes a change in its 3D configuration that
allows binding sites on actin proteins to become exposed [1–
3]. Myosin is now able to bind to this site and hydrolyze ATP to
cause the sarcomere to contract [1–3]. In neurons, activation of
voltage-gated Ca channels causes a sudden increase in the calcium
concentration at the axon terminal [4]. This change prompts the
Yoonjoo Lee and Kristen Segars are co-first authors.

Electronic Supplementary Material: The online version of this chapter (https://doi.org/10.1007/7651_
2020_329) contains supplementary material, which is available to authorized users.
11
12 Yoonjoo K. Lee et al.
release of neurotransmitter-filled vesicles into the synaptic cleft,

propagating the action potential to an adjacent cell [4]. Signaling
through Gq-type GPCRs occurs by cleaving the membrane lipid
phosphatidylinositol biphosphate (PIP2) to inositol triphosphate
(IP3) and diacylglycerol (DAG) upon ligand binding [1]. IP3 binds
to a receptor on the endoplasmic reticulum, causing the release of
stored calcium into the cytosol [1]. This calcium binds to and
activates many different proteins resulting in changes in transcrip-
tion and translation. These are just three examples, but there are
countless other instances where calcium signaling affects cellular
function and cell-cell communication. Given the involvement of
calcium signaling in such a diverse range of processes, having a
reliable way to monitor and analyze calcium mobilization events is
of great interest to researchers in many different fields.
Calcium indicators respond to changes in cytosolic calcium
concentration with changes in their fluorescent properties
[5]. They can be grouped into two main classes: genetically
encoded and chemical [5]. Genetically encoded indicators are
often derived from GFP or its variants fused with calmodulin and
the M13 domain of myosin light chain kinase [5]. These indicators
have the advantage of localization to particular cellular subpopula-
tions and can yield data from in vivo systems such as that following
calcium mobilizations in neurons [5]. These indicators have been
used to map the role of anesthetics on the response of specific
motor neurons in C. elegans [6]. Although extremely useful in
many contexts, we will not be discussing them further in this
chapter.
Like genetically encoded calcium indicators, chemical indica-
tors exhibit substantial changes in fluorescence with increases in
cytosolic calcium concentration [5]. However, there are several key
differences between the two. Whereas genetically encoded indica-
tors are incorporated into an organism’s genome and allow the
organism to produce the fluorescent protein itself, chemical indi-
cators are exogenous substances that are preincubated with ex vivo
or in vitro samples [5] (see Note 1).
The Fura and Indo dyes require UV lasers for excitation and are
ratiometric dyes that demonstrate specific changes in maximum
excitation or emission wavelengths, respectively, upon changes in
intracellular calcium concentration [5, 7]. The Fluo series do not
require UV lasers and exhibit greater fluorescence between bound
and unbound forms, but they do lack the shift in emission spectra
observed in the other probes so that one must carefully decide
which probe to use [7]. The Fluo chemical indicators are based
on the nonfluorescent calcium chelator 1,2-bis(o-aminophenoxy)
ethane-N,N,N0 ,N00 -tetraacetic acid (BAPTA) [7]. BAPTA itself is
structurally similar to the metal ion chelator ethylenediaminetetraa-
cetic acid (EDTA) [7]. BAPTA itself is structurally similar to the
metal ion chelator EDTA but has been modified to yield a higher
Live Cell Imaging 13
affinity for Ca2+ ions and a lower affinity for other metal ions such as
Fe3+ and Mg2+ [7]. The indicators have been modified to optimize
affinity for calcium and yield maximum fluorescence at desired
wavelengths [8]. When calcium binds to an indicator, it undergoes
changes in its 3D structure that greatly enhance its fluorescence, up
to 100-fold in the case of Fluo-3 [7]. In this method discussion, we
are focusing on three of these chemical indicators: Fluo-3 AM,
Fluo-4 AM, and Cal-520 AM.
Fluo-3 AM and Fluo-4 AM are engineered to fluoresce upon
the structural changes induced upon Ca ion binding [5, 7, 8]. Fluo-
4 is a variant of Fluo-3 that has replaced two chlorine atoms with
fluorine [5, 7, 8]. This results in a higher fluorescent excitation at a
wavelength of 488 nm and a higher overall signal [8]. Their maxi-
mum excitation/emission spectra are 506/526 and 494/516,
respectively [8]. Advantages of Fluo-4 include faster loading and a
higher signal at the same concentration [8]. Both Fluo-3 and Fluo-
4 are attached to an acetoxymethyl ester group that blocks the
carboxyl groups on these molecules and facilitates crossing the
plasma membrane [5, 7, 8]. This moiety is cleaved by esterases in
the cell, exposing the carboxyl groups and preventing diffusion out
of the cell [5, 7, 8]. The indicators are denoted as Fluo-3 AM or
Fluo-4 AM if they have this moiety. In addition to Fluo-3 AM and
Fluo-4 AM, there are two additional indicators in the Fluo series:
Fluo-5 AM and Fluo-8 AM [8]. Fluo-5 AM is structurally similar to
Fluo-4 AM but exhibits lesser affinity for calcium ions [7]. This
makes Fluo-5 AM suitable for detecting calcium concentrations
between 1 μM and 1 mM, concentrations that would saturate the
response of Fluo-3 AM or Fluo-4 AM [7]. Fluo-8 AM was engi-
neered to facilitate loading while maintaining the same useful exci-
tation and emission spectra [9]. Whereas Fluo-3 AM and Fluo-4
AM must be loaded at 37 C, Fluo-8 AM can be loaded at room
temperature [9]. Fluo-8 AM is also twice as bright as Fluo-4 AM
and four times as bright as Fluo-3 AM [9]. The Fluo series are
useful for short-term calcium imaging (<1 h) using a 488 nm laser.
One drawback is that they can have high noise (background fluo-
rescence that can hide true positive results and generate false sig-
nals) [10]. There are two main causes for this. The first is that the
bond linking the AM ester to the rest of the molecule can be easily
hydrolyzed in the extracellular space [10]. Therefore, significant
quantities of the dye cannot enter the cells and remains in the
extracellular space where it can still fluoresce [10]. The second
cause is poor localization of the dye to the cytosol due to the activity
of organic anion transporters [10]. These transporters export the
dye into the extracellular space [10]. Initial solutions to this issue
involve co-administering the organic anion transporter inhibitor
probenecid along with the dye [10]. A disadvantage is that proben-
ecid may have effects on the cell that can alter the results and may
cause cytotoxicity [11]. Note that probenecid may also inhibit

channel proteins such as pannexin1 [12].
Cal-520 AM is a newer fluorogenic calcium-sensitive dye with
improved signal and reduced noise relative to the Fluo series of
indicators [10, 11]. In addition there is increased stability of the
AM ester bond that avoids spontaneous hydrolysis in solution and
improves localization and retention in the cytosol [10]. It is rela-
tively unaffected by organic anion transporters, rendering the
co-administration of probenecid unnecessary [10, 11]. It has a
significantly better signal-to-noise ratio compared to Fluo-3 or
Fluo-4 [10, 11]. The increased retention in the cytosol allows for
longer imaging times, of up to 8 h.
Fluo-3 AM, Fluo-4 AM, and Cal-520 AM are all essential tools
for studying the signaling events. In our studies, we have examined
the calcium mobilizations in conjunction with underlying coordi-
nated cell migration in the context of corneal wound healing. When
a cornea is scratched, ATP released from wounded cells causes an
initial burst of calcium mobilization that begins at the wound edge
and is propagated toward the unwounded epithelia [13]. The
release of ATP activates members of the purinergic receptor family
[14, 15]. The initial mobilization is followed by smaller calcium
mobilizations between neighboring cells, which are hypothesized
to be important for coordinating migration and motility [16]. The
use of these tools is critical components of our experimental design.
In addition to calcium indicators, one can use SiR-Actin and
CellMask Far Red dyes to monitor changes in cytoarchitecture.
SiR-Actin was developed from the actin-binding agent jasplakino-
lide. It is fluorogenic and cell-permeable and stains endogenous
F-actin without requiring overexpression in cells. SiR-Actin can be
used for live cell imaging in both tissue and cell cultures. It is stable
for over 18 h when imaging with confocal microscopy or Airyscan
using a low laser light setting. It has maximum excitation and
emission spectra at 652 nm and 674 nm, respectively, comparable
to the spectrum of the far red fluorescent dye CY5. The CellMask
family (Orange, Deep Red and Green) is live stains for the plasma
membrane that internalize slowly compared to other plasma mem-
brane stains such as DiI and labeled wheat germ agglutinin.
CellMask dyes are amphipathic molecules, containing both a
lipophilic moiety that provides excellent membrane loading and a
negatively charged hydrophilic dye for “anchoring” of the probe in
the plasma membrane. They are stable for up to 4 h in live cell
imaging of corneal tissue and cultured cells. This stain generated
from CellMask dyes is maintained after fixation with formaldehyde,
but does not remain when a detergent extraction is used. Excitation
and emission spectra vary depending on the specific dye used.
Options include Green (522 nm/535 nm), Orange (554 nm/
567 nm), and Deep Red (649 nm/666 nm).
These amphipathic molecules have a lipophilic moiety that

provides excellent membrane loading and a negatively charged
hydrophilic dye for “anchoring” of the probe in the plasma mem-
brane. It is stable for up to 4 h in live cell imaging of corneal tissue
and cultured cells. This stain is maintained after fixation with form-
aldehyde, but it does not remain when a detergent extraction
is used.
To understand the distinct roles played by different purinergic
receptors in the initial and later bursts of calcium mobilization and
the cell signaling events responsible for propagating these mobili-
zations between clusters of cells, higher-resolution imaging is
required. The imaging protocols are described below.
2 Materials
Prepare all solutions using Milli-Q water or phosphate-buffered

saline (PBS) pH 7.2 as appropriate. Prepare all reagents the day of
and keep on ice. Reagents such as ATP and UTP are available from
Sigma-Aldrich (St. Louis, MO). Fluo-3 or 4AM fluorescent dye
was purchased from Invitrogen (Carlsbad, CA). CellMask™ Deep
Red Plasma membrane stain was purchased from Thermo Fisher
(Waltham, MA), and SiR-Actin Spirochrome probe was purchased
from Cytoskeleton Inc. (Denver, CO). Cal-520AM was purchased
from ABCAM (USA). Poly(ethylene glycol) diacrylate (PEG-DA),
Irgacure 2959 photoinitiator (2-hydroxy-40 -(2-hydroxyethoxy)-2-
methylpropiophenone), and 3-(trimethoxysilyl)propyl methacry-
late were purchased from Sigma-Aldrich (St. Louis, MO).
3 Methods
3.1 Preparation of 1. Culture cells to desired density for experiment on MAT-TEK

Cells for Ca2+ glass bottom dishes or on tissue culture dishes of different
Mobilization Studies stiffnesses. For flow-through experiments, use a Harvard Appa-
ratus to perfuse fluids.
2. Preload cells with 5 μM Fluo-3, 4 or 5 μM Cal-520AM (Invi-
trogen, Carlsbad, CA), at a final concentration of 1% (v/v)
DMSO and 0.02% (w/v) pluronic acid for 30 min at 37 C
and 5% CO2 (see Note 1).
3. Wash excess dye, and place on stage of microscope in environ-
mental chamber at 35 C and 5% CO2 (see Note 3).
3.2 Ex Vivo In Situ 1. Euthanize rodents according to your institutional IACUC

Preparation of Tissue protocol. Remove the eyes by proptosing them and cutting
for the Study of the optic nerve. Preincubate the eyes/corneas in Fluo-3AM,
Calcium Mobilizations Fluo-4AM, or Cal-520AM to visualize calcium mobilization
for 2 h at 37 C and 5% CO2 (see Note 1). Use the concentra-

tions described in an in vitro setup. Due to the mouse cornea
being a multicell-layered tissue, preincubate with either Cell-
Mask™ Deep Red Plasma membrane stain to visualize cell
membranes for 1 h at 37 C and 5% CO2 or SiR-Actin Spir-
ochrome probe (Cytoskeleton Inc., Denver, CO) to visualize
F-actin for 2 h at 37 C and 5% CO2.
2. Wash the eyes, and induce a corneal injury by causing a scratch
injury on the epithelium, being careful not to injure the base-
ment membrane of the cornea.
3. During the preincubation, prepare MAT-TEK cover glass bot-
tom wells by cleaning with 70% ethanol and allowing them to
air dry. Treat the glass with a solution of 21 mM silane (dilute
3-(trimethoxysilyl)propyl methacrylate into 100% ethanol) by
pipetting an excess on and leaving it for 3 min at room temper-
ature. Rinse the cover glass twice, once with 70% ethanol and
once with water, and allow cover slips to air dry at room
temperature.
4. Prepare the polyethylene glycol solution (dilute 15% polyethyl-
ene glycol with 0.10% Irgacure 2959 photoinitiator
(2-hydroxy-40 -(2-hydroxyethoxy)-2-methylpropipheone in
water).
5. Mount corneas on a salinized glass bottom well. Place the
cornea epithelial side down, and add a drop of polyethylene
glycol gel (PEG).
6. Cross-link the PEG solution in the strata cross-linker at 120 mJ
for 1 min. Make small slits in the agar for penetration of media
using a fire-pulled pipette. Place a cover slip on top of the gel,
and add 1 ml media to the well to prevent sample desiccation.
Place on microscope stage, and image in an environmental
chamber at 35 C that is humidified.
3.3 Imaging 1. Set filters for imaging on instrument of choice. All of these dyes
will work on confocals, SIM, or spinning disks. Fluo-3AM and
Fluo-4AM have a maximal excitation of 506 and a maximal
emission of 526. Cal-520AM has a maximal excitation of
492 nm and a maximal excitation of 514 nm; SiR-Actin has a
maximal excitation of 652 nm and an emission of 674 nm.
CellMask Far Red has a maximal excitation of 650 nm and an
emission of 670 nm. Examples of the use of these indicators can
be seen in Movies 1 and 2.
2. Collect images at the speed required for your experiment.
Users will need to consider the scan speed, the length of time
of imaging, and the memory of the computer. We use a Zeiss
Axiovert LSM 880 confocal microscope and often collect at
3fps for a period of time of up to 4 h. Faster scan times on this
instrument are easily achieved with the FAST module. Higher

resolution is achieved with Airyscan (see Note 2). An example
of imaging tissue with Cal-520AM and CellMask using the
FAST module is shown in Movie 2.
3. Establish baseline values in either control tissue or in cells
before stimulation. We suggest 100–200 frames of baseline
activity so that they represent the population and can be aver-
aged during analysis. Image continually after establishing base-
line values. The Airyscan increases the resolution and overall
image quality compared to traditional confocal microscopy.
4. Save files as LSM and export as AVI for analysis. Movies are
present for viewing on GitHub.
3.4 Analysis 1. Use a customized video processing technique developed to

analyze spatiotemporal communication between groups of
3.4.1 Grid-Based
cells (MATLAB, MathWorks, Inc.). Export videos in TIF or
Modeling of Ca2+
AVI format. Divide the image frame within the video into a
Mobilizations
grid of smaller blocks of equal size with each grid area being
approximately equal to one to two cells. Assign a number for
each block for identification.
2. Average baseline values and subtract from experimental values.
3. Minimize background noise, and compute average intensity.
The grid defining the block-level division will remain fixed over
the entire set of frames within the video, allowing for quantify-
ing inter-block communication and for tracking synchronicity
and behavior of a group of adjacent cells over time.
4. To examine block-level communication over time under differ-
ent experimental scenarios, examine relationships between dif-
ferent blocks to determine if signaling remains in phase.
Compare intensity changes between grid regions, and generate
a comprehensive heat map to display the relationships between
each of the blocks. Determine the average correlation values of
the image studies based on the inter-block correlations values
for each treatment condition. An example of analysis is found in
Fig. 1.
3.4.2 Cell-Based 1. Perform cell-based method analysis to determine the spatio-

Modeling of Ca2+ temporal communication between groups of cells. Export
Mobilizations videos in TIF or AVI format. Use custom MATLAB scripts
(MATLAB, MathWorks, Inc.) to analyze Ca2+ responses based
on a population of cells [17]. Mark cell positions automatically
using a computer program that calculates the X and Y centroid
coordinates of each cell and record them for every registered
fluorescent area from the reference frame.
2. Choose the starting frame. A cell is considered activated when
it exhibits a rapid change in intensity due to the influx of Ca2+
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Title: The curiosities of food

or, The dainties and delicacies of different nations
obtained from the animal kingdom
Author: P. L. Simmonds
Release date: August 28, 2023 [eBook #71509]
Language: English
Original publication: London: Richard Bentley, 1859
Credits: Alan, Aaron Adrignola and the Online Distributed

Proofreading Team at https://www.pgdp.net (This file
was produced from images generously made available
by The Internet Archive)
*** START OF THE PROJECT GUTENBERG EBOOK THE

CURIOSITIES OF FOOD ***
THE
CURIOSITIES OF FOOD;
OR THE
DAINTIES AND DELICACIES OF DIFFERENT
NATIONS
OBTAINED FROM THE
ANIMAL KINGDOM.
BY
PETER LUND SIMMONDS, F.R.G.S., F.S.S.,

AUTHOR OF “A DICTIONARY OF TRADE PRODUCTS,”
“THE COMMERCIAL PRODUCTS OF THE VEGETABLE KINGDOM,”
ETC., ETC.
Cassius. Will you dine with me to-morrow?

Casca. Ay, if I be alive, and your mind hold, and your dinner worth the eating.
Julius Cæsar.
Horatio. O day and night, but this is wondrous strange!
Hamlet. And therefore as a stranger give it welcome.
There are more things in heaven and earth, Horatio,
Than are dreamt of in your philosophy.
Hamlet, Act 1, scene 5.
LONDON:
RICHARD BENTLEY, NEW BURLINGTON STREET.
1859.
LONDON:
PRINTED BY G. PHIPPS, RANELAGH STREET, EATON SQUARE.
P R E FA C E .
The sustentation of the body, and the repairing of its waste by an

adequate supply of wholesome and nutritious daily food, is a subject
of general importance, and necessarily occupies a large share of
attention. But all nations have not the advantages of skilful cattle-
breeders, slaughter-houses, well-supplied meat and poultry markets,
and butchers’ shops graced with all the tempting joints of beef,
mutton, and pork, which gladden the eyes of an Englishman, and
keep up his stamina for labour. The traveller, the settler, and the
savage, must be content to put up with what they can most readily
obtain, and to avail themselves of many an unusual article of food,
which would be rejected under more favourable circumstances, and
with a greater choice for selection.
The subject of Food, in a physiological point of view, has been often
discussed. Popular and learned treatises on all the art and mysteries
of Cookery have been sold by thousands. We have had pleasant
details furnished us too on the Food and the Commissariat of
London.—But with respect to the animal substances, eaten by other
people in foreign countries, we have known little—except from mere
scraps of information.
The basis of the present volume is a lecture on the Curiosities of
Food, which I delivered at several of the metropolitan literary
institutions. Having been favourably received,—from the novelty of
the subject, and the singularity of the specimens from my private
museum by which it was illustrated,—I have been led to believe that
it might prove generally interesting in a more amplified shape.
In order, however, to bring the details within a convenient compass, I
have limited myself to a description of the Curiosities of Animal
Food; but should the work be well received, I may follow it up
hereafter by a companion volume, on the Curiosities of Vegetable
Food.
In the arrangement of the materials for this work, one of two modes
of description was open to me, either to dress up the details in
characteristic pictures of the food-customs and viands in use in
different quarters, and by different people; or to group the whole
scientifically under natural history divisions. As the subject is curious
and striking enough in its simplicity without the aid of fiction or
embellishment, I have preferred adopting the latter arrangement, and
have followed it as closely as the miscellaneous character of the
selections and quotations would permit.
Many of the articles of food named are so outrageously repulsive,
and the consideration of the subject, in a collected form, is altogether
so new, that I have preferred citing authorities in all instances, so as
to relieve myself from the charge of exaggeration, or the imputation
of untenable assertions. For this reason, and from the varied and
very extended nature of the field of enquiry, I can claim no merit for
original writing in this work. I have merely desired to present the
public with a readable volume; and I think its perusal will show that in
this, as in other cases, truth is often stranger than fiction.
After a perusal of these pages, it can scarcely be said that, ‘there is
nothing new under the sun,’ for many of the articles of food which I
have described as being served up in different parts of the world, will
be certainly new to many. Probably, some of the hints thrown out will
make the fortune of any restaurateur in the city or at the west-end,
who chooses to dish up one or more of the reputed delicacies, under
a proper disguise, and with a high-sounding name.
P.L.S.
8, Winchester Street, Pimlico,
December 1858.
CONTENTS.
PRELIMINARY REMARKS.
John Bull’s partiality for Beef, 2. Comparative quantities of meat eaten
in Paris, New York, and London—Different parts of Animals eaten by
choice, 5. Want of courage to taste new Food, 6. Various kinds of
Food eaten in different countries—Man’s omnivorous propensities, 7.
Weight of Food eaten in a man’s life-time—instances of gluttony, 10.
Ethnological view of the question, 11. Jerked beef consumed in Cuba
—Varieties of—tasajo, rebenque, charqui, sesina, 12. Mode of
preparing the sun-dried meat in Chile—Grasa or melted fat, 13.
Biltonge or dried meat of the Cape Colony—Pastoormah—dried
Elephant’s flesh—Hung Beef, 14. Mode of preparing Pemmican, 15.
Gelatine—Beef and Bone Soup, 17. Jellies unnutritious—Portable
Soup, 18. Meat Biscuit—Mode of making it, 19. Utilization of the Blood
of Animals for food, 21. M. Brocchieri’s experiments—Anecdote of an
unlucky Pig doomed to perpetual blood-letting, 23. Arctic luxuries—
Climatic difficulties, 25. A Tuski Feast, 27. A Greenland Banquet, 29.
Animal Food in the Arctic Regions, 30. A Sledge made of frozen
Salmon, 34. Frozen Food brought to the St. Petersburg Market, 35. A
Russian Dining-room, 36. Cooking at Cape Coast Castle—Food
customs and delicacies of the Aborigines of various Countries, 38.
Raw-flesh eaten in Greenland and Abyssinia, 42. Australian Food
delicacies, 45.
QUADRUMANA.
Monkeys eaten in South America, Africa, and the Eastern
Archipelago, 46. Mode of cooking them. Cheiroptera, or hand-winged
Animals—The Fox Monkey, and Bats eaten in the East, 50. Carnivora
—Hyena eaten by Arabs—Pole-cat in North America—Foxes in Italy
—Prairie Wolf in North America, 51. The Lion by the Arabs—The Tiger
by the Malays—The Puma by the Americans, 52. No reason why
Carnivorous Animals should not furnish wholesome and palatable
Food—Bear’s Flesh—A draught of a Quart of Bear’s Grease, 53.
Bear’s Paws and Steaks—Flesh of the Badger, 54. Dogs eaten in
olden times by the Greeks and Romans, and still considered a
delicacy in China, Zanzibar, Australia, and the Pacific, 57. Anecdote of
a Dog Feast. Marsupialia, or Pouched Animals—The Kangaroo—
Food delicacies from it—Mode of cooking, 58. Aboriginal practices
and Food in Australia, 60. Kangaroo-Rat—Opossum—Wombat, 63.
Rodentia—Marmot—Mouse—Musk-Rat, 64. Field-Rat—Rats eaten in
West Indies, Brazil, Australia, China, &c., 65. Chinese Dishes and
Chinese cooking, 66. California bills of fare, 69. Abundance of Rats in
Hong Kong and in Scinde, 62. Salted Rats an article of export from
India to China, 70. Bandicoot, Coffee Rat, Dormouse, Lemming, and
Jerboa eaten as Food, 71. Beaver—Porcupine, 72. Anecdote on
Rabbits, 73. Arctic Hare—Water Dog—Guinea Pig—Agouti—Paca
and Viscascha, 74. Edentata, or Toothless Animals—Native Porcupine
of Australia—Ant-eater and Armadillo, 76.
PACHYDERMATA, OR THICK-SKINNED
ANIMALS.
Baked Elephants’ Paws—Mode of cooking them, 76. Cutting-up the
Elephant, 78. African Haggis—Hippopotamus Flesh and Fat—Zee-
koe Speck, 80. Products of the Hog—Reading Bacon and eating
Bacon, 81. Swine feeding on Corpses in the Ganges, 82. Pigs fed on
Mutton, 83. Acres of Pork in America, 84. ‘Going the whole Hog,’ 85.
Origin of roast Pig, 86. Spanish Pigs, 90. Toucinho, or fat Pork, used
in Brazil—Peccary, Rhinoceros, and Tapir eaten, 92. Horse-flesh, the
recent endeavours to popularize it as an article of Food, 94. M. St.
Hilaire’s exertions in the cause—Historical progress—Horse-flesh
eaten in Africa, America, Asia, and Europe, 97. Experimental trials
and cooking, 100. Horse-flesh eaten unknowingly in many cases, 104.
Anecdote of Sausages—Evidence before Parliamentary committee
respecting Horse Sausages, 105. Unwholesome Meat, 106. Blowing
Veal, 109. Asses’ Flesh—The Quagga 110.
RUMINANTIA AND CETACEA.

Camel’s Flesh, 111. Axis Deer—Moose Deer—Caribboo—Venison not
Meat in North America—Reindeer, 112. Giraffe—Eland—Hottentot
cooking—Antelope Tribe—The Hartebeest—Sassaby—Ourebi—
Boshbok—Rheebok—Gnu, &c., 113. Alpaca Tribe—Sheep’s Milk—
Large Tailed Cape Sheep—Dried Flesh of the Argali—Goat’s Flesh,
115. Bison Beef—Buffalo Humps—Musk-Ox, 116. Cetacea—Manatus,
117. Flesh and Tongue of the Sea-Lion, 118. Walrus Meat—Sea-Bear
—Seal Flesh, 119. Flesh of the Whale eaten in various quarters—
Porpoise, an ancient dainty—Mode of serving it at the tables of
English Nobility, 120.
BIRDS.
No Carnivorous Birds eaten—Insessores or Perching Birds—Becafico
—Edible nest of the Eastern Swallow or Swift, 122. Mode of collecting,
localities, statistics, and details in the Eastern Archipelago, 123. The
Guacharo Bird, 128. The Diablotin or Goat-sucker—Spitted Larks,
129. Crows, Thrushes, and Robin Redbreasts eaten in Italy—The
Rice Bunting, 130. The Toucan—Parrot Pie—Gallinaceous Fowls—
Peacock Enkakyll, 131. Wild Turkey of New Granada—Value of
Poultry and Eggs consumed, 132. Fixed Tariff for Poultry and Game,
&c., in London in 1272—Price of Eggs, Pigeons, &c., in 1313, 133.
Prices of Poultry and Game in 1575, 134. Prices of Food and Poultry
in 1531, 135. Ancient Receipt for making a Game Pie in 1394, from
the Books of the Salter’s Company—Prices of Cattle and Dairy
produce in 1548, 137. Consumption and Statistics of Eggs—
Comparative use in Paris and London—Imports from Ireland—Modes
of testing the quality of Eggs, 138. Preservation of Eggs—Salted Eggs
—Pickled Eggs—Painted Eggs—Condensed Egg, 140. Roman
Preserves for fattening Poultry—Wild Game in Jamaica, 142.
Canadian mode of cooking Partridge, 143. Red-legged Partridge run
down on foot—Quail—Turtle Dove—Passenger Pigeon, 144. Hogs fed
upon the Squabs—Canvas-back Duck, 146. Cock of the wood—Wild
Birds of New Zealand, 147.
GRALLATORES.
Ostrich and Emu Eggs, 148. Bustards, 149. Clucking Hen and
Mangrove Hen of Jamaica, 150. Bittern—Snipe Woodcock—
Flamingoes’ tongues, 152.
NATATORES.
Sea-gulls eaten by the Chinese—Livers and Hearts of Penguins—
Puffins pickled with Spices, 153. The Mutton-Bird of Australia—Habits
of the Bird—Mode of taking them by the New Zealanders, 154. Birds
eaten in the Arctic Regions—Grouse Pie—Dovekey and Auk Pie—
Guillemot Soup, 156. Eggs of Sea Fowl—Large sale of them in San
Francisco, and at the Cape of Good Hope, 158. Penguins’ Eggs in
Tristan d’Acunha, 160. The Rookeries—Exciting Sport, 161. Annual
Egg gathering visits to the Pedro Keys from Jamaica—Description of
the Islets—Birds which frequent them—Recognised customs among
the Boatmen—The Egg Bird, 163. Turtle Eggs, 167. Wild and
Domestic Geese—Half-hatched Eggs eaten by the Esquimaux, 168.
Cygnets—Pintail Duck—Widgeon and Teal, 169.
REPTILIA.
Enumeration of the Reptiles in the Four Orders, eaten as Food, 169.
Land Tortoises and their Eggs, 170. Terrapin or Box Tortoise—Cruel
mode of killing them, 171. Tenacity of Life—Fluviatile Tortoises—The
Hiccatee of Honduras, 172. Shooting a Turtle—Abundance of large
Land Tortoises in the Gallipagos Islands—Very generally eaten in the
Pacific, Australia, South America, and Europe—Tortoise oil, 174.
Salted Turtle—Chasing the Turtle—Horrible process of removing the
Shell, 175. Dampier’s Description of Land Tortoises in the West Indies
in 1684—First Introduction of Turtle to England—Statistics of
Consumption—Noted City Houses for Turtle Soup, 176. Turtling in the
Grand Caymans, West Indies, 177. Mock Turtle and Real Turtle—
Ascension the Head Quarters for keeping Turtle, 178. Adventures of
Old ‘Nelson’—Turtle should be sent home in a Sealed Cask—Jaguars
of South America fond of Turtle and their Eggs—A Brazil Native will
eat 20 or 30 Turtle Eggs at a meal, 180. Description of the Eggs—
Hawk’s-bill Turtle eaten, but sometimes unwholesome—Collecting
Turtle Eggs on the Orinoco, by the Indians—Preparation of an Oil
called ‘Mantega’ from them—Gives Employment to several thousand
Persons, 181. Quantity made and Value—Not very pure—Uses of
Turtle Oil for Culinary and Illuminating Purposes, 182. The Iguana—
Description of it—Repulsive Appearance—Very Delicate Eating, 183.
Mode of cooking it—First Repugnance of the Early Spaniards to it, as
related by Peter Martyn, 184. Mode of Catching the Reptile by
Natives, 185. Hunted by Dogs in the Bahamas Islands—Met with and
esteemed in Australia, 186. Aboriginal Appreciation of it—Eaten by
Natives of Ceylon, 187. Eggs of this Lizard an esteemed Delicacy—
Should be introduced to our Tables—All kinds of Lizards eaten by the
Blacks of Australia, 188. Lizard Family obnoxious to Poisons—Lizards
brought to the Rio Janeiro market—Hatching a Crocodile by a Fancy
Poultry Breeder, 190. Eggs of the Alligator eaten—Effect of
Imagination on the Stomach, at a Dinner given by Dr. Buckland, 191.
Australian Crocodile eats like Veal, 192. Origin of the Australian
‘Bunyip’ Fiction—Flesh of the Crocodile musky, 193. Various Opinions
of Alligator Meat, 194. An Alligator Hunt in South America, 195. Eggs
and Skin of the Alligator eaten—Oil prepared from the Fat, 196.
Lizards, Serpents, and Snakes, 197. Swallowing Live Lizards
supposed to cure the Cancer—Boa-constrictor eaten, 198. Fried
Rattlesnake or ‘Musical Jack,’ 199. Roasted Snakes in Australia, 202.
Extending use of Frogs for Food in Europe, America, and the East—
Toads frequently sold for frogs, 204. Mode of skinning and preparing
them—Eaten boiled in Brazil, without any Preparation, 206.
FISH.
Abundance of Fish—Modes of preserving them—Analyses of their
Flesh, 208. Presence of Iodine, 210—Fish Chowder—Fish Glue and
Isinglass—Fish-maws, immense Trade in, 211. Caviar and the dried
Roes of Fish, 212. Ancient Customs, Prices, and Kinds of Fish used,
215. Fish Ordinaries, 216. The Russian Piroga, an oily Fish-cake—
Dried loaves of putrid pounded Fish eaten in Africa and South
America, 218. Bony Fishes—Unwholesome and Poisonous Fishes—
Assumed Causes for the Fish Poison, 219. Fish Liver and Gall, 221.
Classification of Fishes—Neglect of our Fisheries, 222. Ocean Fishes
dry eating—Mode of drying the Bonito—The hard horny pieces, under
the name of Cummelmums, used to rasp over Rice, 223. Shark’s
Flesh sold in the Havana Market—Shark Hunting—Excitement of the
Sport, 224. The Picked Shark—Spotted Dog-Fish—Pigs fed on them
—Shark oil, 228. Fisheries for the Sharks in India for the Fins—
Extensive Trade in these to China—Dogs trained to bring Sharks
ashore, 229. Anecdotes of Sharks, 231. The Sturgeon, a Royal Fish—
Flesh not much esteemed—Sturgeon’s Skull-cap and Shark’s Fin
Stew, Chinese Delicacies, 236. Lampreys—Eel Pies and stewed Eels
—Spearing Eels—Jews prohibited from eating them, 238. Comparison
between British Fish and Mediterranean Fish, 240. Finnon Haddock—
Fresh Herrings—Pickled Herrings—Red Herrings and Bloaters—
Origin of Smoked Herrings—Herring Pies sent from Yarmouth
periodically to the Queen, 242. Conger Eel dried and grated to powder
for making Fish Soup—Congers formerly reared in Vivaria by the
Romans, 244. The Sand-Eel and Sand-launce—Smelts—Whitebait,
245. Substitutes for Whitebait in distant Seas, 246. The Anchovy—An
Irishman’s Blunder, 249. The Sardine Fishery, 252. West Indian
Fishes—Hog Fish—Snapper—Queen Mullet—Paracuta—Callipeva—
Red Mullet—King Fish, &c., 253. The Sun Fish—Pacou—Gourami—
Caffum, 256. The Pirarucu—The Sheep’s Head—The Green Cavalla
—The John and Goggle-Eye—The Flying Fish, 259. Sprats—
Coveeching Fish—Mud Fish, 261. Lut-fisk of Sweden—Fish exported
from New Brunswick—The Sea-perch—The striped Bass—Brook
Trout and Sea Trout, 263. Gaspereaux or Alewives—Salmon-trout—
Skate—Capelan—Halibut Fins—Smoked Eels in New Brunswick and
Port Phillip, 266. White Fish of the North American Lakes—Gizzard
Fish—Mashkilonge—Trout and other Lake Fish, 268. Modes of
Fishing—Scoop Nets and Gill Nets—Angling through the Ice, 271.
Extreme Fatness of Lake Fish, 274. Fish Soup, 275. Fish of the
Pacific Coasts—Robalo, Corvino, Lisa, Bagre, 227. A Hawaiian
Restaurant—Raw Fish eaten—Salmon the King of Fresh-water Fish,
278. Salmon Fisheries of Oregon and California, 279. Chinese
Fisheries—Fish of the Australian and Indian Seas—Tamarind-fish—
Mango-fish—Black and white Pomfrets—Bombay Duck, 284. Fish of
the Cape Colony—Géelbeck or Cape Salmon, Snook, Silver fish,
Harders, Jacob Evertsen, Kabeljauw, Hottentot Fish, Windtoy,
Bamboo Fish, Galleon, Lake, &c., 286.
INSECTS.
Insects furnish many good Delicacies—Fairy Cates, 292. Coleoptera
—Larvæ or Grubs of Beetles eaten in various localities—Roman
Epicures used to Fatten them, 293. Goliath Beetles eaten in Africa—
Turkish Women cook Beetles in Butter to fatten themselves, 295.
Orthoptera—Locusts extensively eaten in Africa and Arabia—Modes
of Collecting and Cooking them, 296. Animals and Birds feed greedily
on them—Descriptions given by various Travellers, 300. Locusts
eaten in Eastern Asia—Grasshoppers tried and found to be good
eating—A Grasshopper Roast in California, 304. Neuroptera—
Termites, or White Ants, eaten by the Africans and South American
Indians—Yellow and Red Ants in Brazil—Ants Distilled for Brandy in
Sweden—Cocoons of the Wood Ant collected and sold for Feeding
Birds, 305. Caviar of Insect Eggs in Mexico—Axayacat—Mode of
Collecting—Cakes and Bread, called Hautle, made from them—Curry
of Ants’ Eggs, 306. Hymenoptera—Bees eaten in Ceylon—
Caterpillars of the Butterfly—Silk-worm Chrysalids Bugong Moth, a
great Delicacy to Natives of Australia—Sage-Apples or Galls, in the
Levant, 311. Hemiptera—The Cicada or Chirping Flies eaten in
America and Australia—Caterpillars eaten like Sugar Plums, 314.
ARACHNIDA.
Spiders eaten in Various Quarters as Centipedes are in others, 316.
CRUSTACEA.
Flesh of Crustaceans Difficult of Digestion—Varieties of Consumed—
Land Crabs—Their Habits—Varieties—Mode of Cooking them—An
Ingredient in the Famous ‘Pepper-pot,’ 316. Abundance of Land Crabs
at the Bahamas—mentioned by Virgil—Mason Crab of Chile eaten,
321. The Lobster—Where principally Caught—Preserved Fresh
Lobsters, 322. Salted Lobsters—Pond or Saltern, for keeping them, at
Southampton—A Tale with a Moral, 327. Turning Lobsters on their
Backs, 328. Live Crablets eaten by the Chinese, 329. Shrimps and
Prawns—Enormous Consumption of them—Instructions for Cooking
them, 350. Dried Prawns and Shell-fish—Large Trade in them in the
East, 332. Balachong or Gnapee, 333.
MOLLUSCA, &c.
Oysters—‘Natives and Scuttlemouths’—Racoon or Parasitic Oysters,
334. Large Trade in Oysters in America, at New York, Baltimore,
Boston, and New Orleans, 335. Bottled Oysters at the Cape—Mussels
—‘Old Maids’—Scallops—Clams and Clam Digging—Largely used for
Bait, 342. Periwinkles—Large Consumption of, in London—Whelks,
Boiled and Pickled, 345. Snails a Fashionable Article of Diet—Roman
Taste for them—A Snail Pie—The Vineyard Snail—Modes of Dressing
them, 346. Attempt of Two Philosophers to relish them, 347. Snail
Soup—The Parrot’s-bill Barnacle eaten, 349. Annelida—Palolo, a
Pacific Delicacy, 350. Diet of Worms—Cuttle-fish eaten, 352. Arcas
and Monodonta eaten—Sea Eggs or Urchins, 353. Holothuria—the
Sea Slug Soup of the Chinese—Bêche-de-mer or Tripang, 354. The
Times’ Correspondent’s Opinion of this Dish, 355. Extensive Fisheries
for the Animal, 356. Details of the Preparation and Statistics of the
Trade, 358. Varieties and Prices, 364.
CONCLUDING REMARKS.
Ignorance as to some of our Common Food—Ox Tongues—Polonies
—Confidence inspired by the Pie-man eating one of his own Pies,
367. We eat many things which would be refused by others, 368.
Bounty and Wisdom of the Creator in providing for Man—Difficulty of
determining what are Food Delicacies, 369. New Varieties of Food
may be Provided Artificially—Fresh Hides of Cattle a Delicacy in Java
—Buffalo Hides and other Skins made into Jellies at Home—Buckskin
Breeches, boiled and stuffed with Sea-weed for Food—Resumé of the
Dainties of Different People—Verification of the Proverb—‘One half
the world does not know how the other half lives.’
THE
CURIOSITIES OF FOOD.
What is the prevailing food of the people? Is it chiefly animal or

vegetable, and whence is it derived in the two kingdoms? Do they
trust to what the bounty of Nature provides, or have they the means
of modifying or controlling production, whether in the cultivation of
vegetables, or the rearing of animals? Describe their modes of
cooking, and state the kinds of condiments they employ. Have they
in use any kind of fermented liquor? What number of meals do they
make, and what is their capacity for temporary or sustained
exertion?
These are some of the enquiries to which a traveller is directed to
pay attention, if he wishes to furnish and diffuse useful information.
I do not intend to go over this wide field of investigation in the
systematic and scientific manner shadowed forth by these enquiries,
but merely desire to assist the reader to pass a leisure hour,
although he may probably glean some useful information at the
same time.
I propose bringing under notice some of the Animal food in which
people in various countries indulge, not that I wish persons to test
these meats, or to live upon them, unless they please. I do not deal
in them, and have no interest in their collection or sale, but I merely
desire to introduce them to notice that the reader may ascertain the
opinions entertained of them, think over them, and know how much
better an Englishman is fed than any one else in the world. So that,
despite our habit of grumbling, there is at least this undeniable fact
before us, that the middle classes are in very easy circumstances;
and that English workmen earn good wages, or they could not
consume the quantity of animal food they do at the present prices.
According to Vauban, Bossuet, and La Grange, the richest and most
comfortable nation is that which eats the most meat. At the present
prices of this article here, it certainly must be so, for a poor nation
could not indulge in the luxury.
Beef and mutton, and mutton and beef, no matter what their price,
John Bull will not dispense with; and although they are 40 or 50 per
cent. dearer now than they were ten years ago, and although we
import animals largely from abroad, and our cattle-breeders do their
best to meet the demand, cattle and sheep will not increase and
multiply fast enough to bring down the price for the consumer.
A writer in Household Words thus alludes to our national weakness.
—‘Next to the Habeas Corpus and the Freedom of the Press, there
are few things that the English people have a greater respect for and
a livelier faith in than beef. They bear, year after year, with the same
interminable, unvarying series of woodcuts of fat oxen in the
columns of the illustrated newspapers; they are never tired of
crowding to the Smithfield Club cattle-show; and I am inclined to
think that it is their honest reverence for beef that has induced them
to support so long the obstruction and endangerment of the
thoroughfares of the metropolis by oxen driven to slaughter. Beef is a
great connecting link and bond of better feeling between the great
classes of the commonwealth. Do not Dukes hob and nob with top-
booted farmers over the respective merits of short-horns and
Alderneys? Does not the noble Marquis of Argentfork give an ox to
be roasted whole on the village green when his son, the noble
Viscount Silvercoral, comes of age? Beef makes boys into men.
Beef nerves our navvies. The bowmen who won Cressy and
Agincourt were beef-fed, and had there been more and better beef in
the Crimea a year or two ago, our soldiers would have borne up
better under the horrors of a Chersonesean winter. We feast on beef
at the great Christian festival. A baron of beef at the same time is
enthroned in St. George’s Hall, in Windsor’s ancient castle, and is
borne in by lacqueys in scarlet and gold. Charles the Second
knighted a loin of beef, and I have a shrewd suspicion that the
famous Sir Bevis of Southampton was but an ardent admirer and
doughty knight-errant in the cause of beef. And who does not know
the tradition that even as the first words of the new-born Gargantua
were ‘A boyre, à boyre,’ signifying that he desired a draught of
Burgundy wine—so the first intelligible sounds that the infant Guy of
Warwick ever spake were ‘Beef, beef!’ When the weary pilgrim
reaches the beloved shores of England after a long absence, what
first does he remark—after the incivility of the custom-house officers
—but the great tankard of stout and the noble round of cold beef in
the coffee-room of the hotel? He does not cry ‘Io Bacche! Evöe
Bacche!’ because beef is not Bacchus. He does not fall down and
kiss his native soil, because the hotel carpet is somewhat dusty, and
the action would be, besides, egregious; but he looks at the beef,
and his eyes filling with tears, a corresponding humidity takes place
in his mouth; he kisses the beef; he is so fond of it that he could eat
it all up; and he does ordinarily devour so much of it to his breakfast,
that the thoughtful waiter gazes at him, and murmurs to his napkin,
‘This man is either a cannibal or a pilgrim grey who has not seen
Albion for many years.’
It has been well observed, that there are few things in which the
public have so great and general an interest, and concerning which
they possess so little real knowledge, as of the provision trade and
the wholesale traffic in animals live and dead, in their own and other
countries. When, where, and how raised, and what processes meat
passes through before it reaches their tables, are questions which,
though highly important, are very seldom asked by the consumers—
all that they usually trouble themselves with is, the current retail
price, and the nature of the supply.
Few of us think as we sit down to our rump steak or pork chop, our
sirloin or leg of mutton, of the awful havoc of quadrupeds necessary
to furnish the daily meals of the millions. I will not weary the reader
with statistics, although I have a long array of figures before me,
bearing upon the slaughter of animals for food in different countries.
It will be sufficient to generalize.
If the hecatomb of animals we have each consumed in the years we
have lived, were marshalled in array before us, we should stand
aghast at the possibility of our ever having devoured the quantity of
animal food, and sacrificed for our daily meals the goodly number of
well-fed quadrupeds of the ovine, bovine, and porcine races, or the
fish, fowl, reptiles, and insects, which would be thus re-embodied.
The average quantity of animal food of all kinds consumed in France
is stated on good authority—that of M. Payen—to be as low as one-
sixth of a pound per diem to each person. Even in the cities and
large towns, especially Paris, the amount of food upon which a
Frenchman lives is astonishingly small. An Englishman or an
American would starve upon such fare.
In proportion to its population, New York consumes as nearly as
possible the same quantity of meat as London, about half-a-pound a
day to each person; more beef, however, is consumed there and
less mutton, and the latter fact may be accounted for by the
comparative inferiority of quality.
It is curious to notice the various parts of animals that are eaten, or
selected as choice morsels by different persons or classes. Sheep’s
head, pig’s head, calf’s head and brains, ox head, the heads of
ducks and geese, ox tongue, reindeer tongue, walrus tongue,
crane’s tongue, &c. Fowls and ducks’ tongues are esteemed an
exquisite Chinese dainty. The pettitoes of the sucking pig, or the
mature feet and hocks of the elder hog, sheep’s trotters, calf’s feet,
cow heel, bear’s paws, elephant’s feet, the feet of ducks and geese,
and their giblets; ox tail, pig’s tail, sheep’s tail, kangaroo tail,
beaver’s tail. And the entrails again are not despised, whether it be
bullock’s heart or sheep’s heart, liver and lights, lamb’s fry or pig’s
fry, tripe and chitterlings, goose liver and gizzard, the cleaned gut for
our sausages, the fish maws, cod liver, and so on. The moufle, or
loose covering of the nose, of the great moose deer or elk is
considered by New Brunswick epicures a great dainty. The hump of
the buffalo, the trunk of the elephant, are other delicacies. Deer’s
sinews, and the muscle of the ox, the buffalo, and the wild hog,
jerked or dried in the sun, and then termed, ‘dendeng,’ is a delicacy
of the Chinese, imported at a high price from Siam and the eastern
islands.
The eggs of different animals, again, form choice articles of food,
whether they be those of the ordinary domestic poultry, the eggs of
sea fowl, of the plover, and of game birds, of the ostrich and emu, of
the tortoises and other reptilia, as alligator’s eggs, snake’s eggs, and
those of the iguana, or the eggs of insects, and of fishes.

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Stem Cell Renewal and Cell Cell

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Ottawa, ON, Canada Kursad Turksen

Inference of Ligand–Receptor Pairs from Single-Cell Transcriptomics Data. . . . . . . . . 1

Promoter Pull-Down Assay: A Biochemical Screen for DNA-Binding

MATTHEW ANTEL • Department of Cell Biology, University of Connecticut Health Center,

WILLIAM J. KOCH • Department of Biomedical Sciences Heritage College of Osteopathic

Madrid, Spain; Vascular Pathophysiology Area, Centro Nacional Investigaciones

MASAYUKI YAMASHITA • Center for Basic Medical Research, International University of

Inference of Ligand–Receptor Pairs from Single-Cell

Key words Cell–cell communication, Single-cell RNA sequencing, Receptors, Ligands

Intercellular communication underlies numerous biological pro-

calculate which ligand–receptor pairs display significant cell-type

CellPhoneDB can be used either through the interactive website

2.2 Software – Python 3.5 or higher.

2.3 Hardware – Linux or MAC OS.

3.1 Installation We recommend using a virtual environment, but this is optional

--project-name: Project’s name. A subfolder with this identity

l A usage example to set output path, number of iterations, and

In addition to the parameters described in step 3, there are also

Dot plot specific parameters:

To plot only specific rows/columns, use text files with a list

2. For heatmap visualization, run the heatmap visualization com-

Specific parameters for the heatmap plot:

--pvalues-path: The pvalues output file.

The –database <version_or_file> parameter can be either a

3. List available versions from the local repository, stored locally

4. Download a specific version from the remote repository.

version_spec must be one of the database versions in the

cellphonedb database generate

Database generate specific parameters:

--user-protein: Protein input file

The database file will be generated in the --result-path folder

cellphonedb method statistical_analysis

l Use only user-specific interactions without merging with the

l Add or modify some existing interactions (using custom_inter-

For duplicated interactions, the user specified interactions

cellphonedb database generate --user-protein

l Update database from remote servers, for example from

cellphonedb database generate –fetch

cellphonedb method statistical_analysis metafile.txt

1. CellPhoneDB stores ligand–receptor interactions and informa-

interaction pairs stored in the database as proteins. Specifically,

6. CellPhoneDB users can also generate their own cell–cell inter-

We thank Sarah Teichmann for useful guidance on the method and

Multiple Imaging Modalities for Cell-Cell Communication