Advanced Methods in Cellular Immunology - R, Bio Med 1
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Advanced Methods in
Cellular
Immunology
Rafael Fernandez-Botran
Václav Vetvicka
Advanced Methods in
CELLULAR
IMMUNOLOGY
Rafael Fernandez-Botran, Ph.D.
Václav Vetvicka, Ph.D.
CRC Press
Boca Raton London New York Washington, D.C.
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Preface
Immunologists, as well as investigators in other disciplines, often need to
use protocols involving the isolation, culture, or characterization of different
types of leukocytes; they may need to study cytokine expression by different
cells or use a variety of animal models of inflammation and/or autoimmu-
nity. With the same idea as that of our previous book,
Methods in Cellular
Immunology
, we have compiled in this book techniques in the above areas
and presented them in an easy-to-use format. Each chapter provides readers
with related background information and a step-by-step description of the
methodology, alternative techniques, pertinent references, and information
about commercial sources for materials and reagents.
This book focuses on the isolation and functional characterization of
different types of leukocytes, cell culture, and different animal models of
inflammation and autoimmunity. Chapter 1 covers the preparation of cell
suspensions from several sources, including bone marrow, thymus, and sec-
ondary lymphoid organs, together with culture techniques and protocols to
measure cell proliferation. Chapters 2 and 3 are dedicated to the isolation
and functional assessment of neutrophils and eosinophils, respectively, in-
cluding techniques based on density-gradient centrifugation and the more
novel immunomagnetic bead isolation protocols. Chapter 4 describes the
isolation of human basophils. Chapter 5 examines the isolation and functional
characterization of monocytic cells, including assays for the production of
oxidative metabolism intermediates, phagocytosis, pinocytosis and bacteri-
cidal/antiviral activities. Chapter 6 focuses on B lymphocyte cloning and
culture methods, and Chapter 7 explains general techniques in cell culture.
Chapter 8 describes the techniques of reverse transcriptase polymerase chain
reaction (RT-PCR) and ribonuclease protection assay (RPA), and their appli-
cations in the study of cytokine mRNA expression. Chapters 9 and 10 describe
different techniques for the intracellular detection of cytokines and the mea-
surement of cell cytotoxicity and apoptosis. Chapter 11 explains mixed lym-
phocyte reaction. Chapter 12 outlines animal models of autoimmune diseases.
Chapter 13 explains the preparation of several common reagents. Chapter 14
provides a list of manufacturers/commercial sources, including addresses,
telephone numbers, and Web sites.
The Authors
Rafael Fernandez-Botran, Ph.D.,
is an associate professor in the Department
of Pathology and Laboratory Medicine, Division of Experimental Immunol-
ogy and Immunopathology, School of Medicine, University of Louisville,
Kentucky. Dr. Fernandez-Botran received his bachelor of science degree in
biological chemistry from San Carlos University, Guatemala in 1979 and his
Ph.D. in microbiology from the University of Kansas Medical Center, Kansas
City in 1985. Dr. Fernandez-Botran worked as a postdoctoral fellow and
instructor in the laboratories of Dr. Ellen S. Vitetta and Dr. Jonathan W. Uhr
at the Department of Microbiology, University of Texas Southwestern Med-
ical Center, Dallas, where his work centered on the study of cytokines, par-
ticularly interleukin-4, and their roles in the proliferation and differentiation
of CD4
T cell subsets and B cells, and on the characterization of interleukin-
4 receptors and their interactions with interleukin-2 receptors.
More recently, Dr. Fernandez-Botran’s major research interests have fo-
cused on the immunoregulatory roles of soluble cytokine receptors and the
interactions between cytokines and glycosaminoglycans. Dr. Fernandez-Bot-
ran has received several fellowships and awards, including the New Inves-
tigator Recognition Award from the Clinical Immunology Society (CIS) in
1989, and the President’s Young Investigator Award from the University of
Louisville in 1993. His research activities have resulted in more than 50
published works, including articles, several reviews, and chapters.
+
Václav V ˇtviˇka,
is an assistant professor in the Department of
Pathology, Division of Experimental Immunology and Immunopathology,
School of Medicine, University of Louisville, Kentucky. Dr. grad-
uated in 1978 from Charles University in Prague, Czech Republic with an
advanced degree in biology. He obtained his Ph.D. in 1983 from the Czech-
oslovak Academy of Sciences, Institute of Microbiology, Prague. He is a
member of the Czech Immunological Society, American Association of Im-
munologists, International Society of Developmental and Comparative Im-
munology, and American Association for Cancer Research. In 1984 he was
awarded the Distinguished Young Scientist Award of the Czechoslovak
Academy of Sciences. During 1984 to 1985 and 1988 he spent a total of 18
months as a research associate at the Oklahoma Medical Research Founda-
tion, Oklahoma City.
Ph.D.,
V
ˇ
tvi
ˇ
ka
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