Cytokine Bioassays: Methods and Protocols

367 Pages·2014·8.826 MB·English

by  Ivana Vancurova (eds.)

Methods in
Molecular Biology 1172
Ivana Vancurova Editor
Cytokine
Bioassays
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Cytokine Bioassays
Methods and Protocols
Edited by
Ivana Vancurova
Department of Biological Sciences, St. John’s University, Queens, NY, USA
Editor
Ivana V ancurova
Department of Biological Sciences
St. John’s University
Queens, NY , USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-0927-8 ISBN 978-1-4939-0928-5 (eBook)
DOI 10.1007/978-1-4939-0928-5
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Prefa ce
Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes
and associated with a wide range of diseases, including immune and infl ammatory disor-
ders, as well as many types of cancer and leukemia. Examples of cytokines include interleu-
kins, chemokines, growth factors, interferons, and the tumor necrosis factor family.
Information about the qualitative and quantitative nature of cytokine expression and release
is essential for the understanding of physiological and pathological processes. However, the
cytokine detection in biological and clinical samples faces many challenges that include
the low abundance, the need to distinguish between active and latent cytokine forms,
and the need to measure multiple cytokines in a single assay. This volume provides a compre-
hensive collection of classic and cutting-edge methodologies that are used to analyze and
quantify cytokines and their biological activities in complex biological and clinical samples.
The chapters are divided into three main categories. The fi rst category focuses on the
immunodetection of released cytokines in tissue culture supernatants, plasma, serum, and whole
blood samples by immunoassays. These immunoassays measure the total concentrations of
released cytokines regardless of their biological activities, and include ELISPOT, immunob-
lotting, and ELISA. In addition, the complexity of cytokine responses has led to the develop-
ment of multiplex technologies that can simultaneously detect large numbers of cytokines in
small sample volumes. Thus, the fi rst part also includes the recently developed multiplex
arrays that allow the simultaneous measurement of multiple cytokines in small sample vol-
umes with time-saving advantages and less cost compared to other immunoassays.
Since immunoassays cannot distinguish between biologically active and inactive cyto-
kines, bioassays are used to measure different biological activities of cytokines, such as
cytokine-i nduced cytokine release, chemotaxis, phagocytosis, proteasome activity, and
immunoglobulin class switching. The second part focuses on the analysis of biologically
active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-
induced phagocytosis assay, proteasome activity assay, and analysis of cytokine-induced
immunoglobulin class switching. In addition, since cytokines exert their functions through
binding to cytokine receptors, this category also includes methods focused on the analysis
of expression and function of cytokine receptors.
Often, there is a need to identify the cytokine-producing cells, or to analyze the intra-
cellular cytokine protein levels or the mechanisms regulating cytokine expression. The t hird
part focuses on the a nalysis of intracellular cytokines by fl ow cytometry, immunohistochem-
istry, immunofl uorescence confocal microscopy, and western blotting. In addition, this cat-
egory includes protocols for quantitative a nalysis of cytokine gene expression by real-time
RT-PCR, luciferase assay, analysis of the cytokine promoter occupancy by chromatin immu-
noprecipitation, and analysis of alternative splicing of cytokine genes.
There is no one best method, since each has its own merits and limitations. Choosing
the right method depends on the sample, purpose of the assay, and the available instrumen-
tation. Often, using combined information from several different approaches can yield the
most accurate information about the quantitative and qualitative nature of cytokine expres-
sion. The reliability of all the protocols has been tested in laboratories around the world.
v
vi Preface
Each chapter is appended by notes that navigate through the protocol and serve as a
troubleshooting guide. By covering a broad variety of methods used in cytokine research
and analysis, we hope that this book will be useful not only to biochemists, molecular biolo-
gists, and immunologists, but also to physician-scientists working in the fi eld of cytokine
research.
I would like to thank all the authors for their enthusiastic help and support in assem-
bling this volume; I fully realize that in the highly competitive environment of academic
research, many scientists are reluctant to commit their time to writing book chapters and
method articles. I also would like to express my gratitude to the series editor, Dr. John
Walker, and the outstanding staff of Humana Press for their support, help, and
encouragement.
Queens, NY Ivana V ancurova
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
PART I DETECTION OF RELEASED CYTOKINES BY IMMUNOASSAYS
1 The Challenge of Measuring Elusive Immune Markers
by Enzyme-Linked Immuno-Spot (ELISPOT) Technique. . . . . . . . . . . . . . . . 3
Maria F aresjö
2 Analysis of the Released Nuclear Cytokine HMGB1 in Human Serum . . . . . . 1 3
Haichao Wang, L in Z hao, J ianhua L i, S hu Z hu, and Maggie Yeung
3 ELISA-Based Assay for IP-10 Detection from Filter Paper Samples. . . . . . . . . 2 7
Camilla Heldbjerg Drabe, T homas B lauenfeldt, and M orten Ruhwald
4 E nhanced ELISA Based on Carboxymethylated Dextran Coatings. . . . . . . . . . 3 9
Benoît L iberelle, C harles F ortier, and Gregory De Crescenzo
5 A Novel Three-Dimensional Biosensor Based on Aluminum Oxide:
Application for Early-Stage Detection of Human Interleukin-10 . . . . . . . . . . . 4 9
Michael Lee, A bdoullatif B araket, N adia Z ine, Miguel Z abala,
Francesca Campabadal, Raffaele C aruso, Maria G iovanna T rivella,
Nicole Jaffrezic-Renault, and Abdelhamid Errachid
6 Quantification of Multiple Cytokines and Chemokines
Using Cytometric Bead Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Gemma Moncunill, J oseph J . C ampo, and Carlota D obaño
7 A Useful Guide for Analysis of Immune Markers
by Fluorochrome (Luminex) Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Maria Faresjö
PART II CYTOKINE BIOASSAYS
8 Control of Pro-inflammatory Cytokine Release from Human
Monocytes with the Use of an Interleukin-10 Monoclonal Antibody. . . . . . . . 99
Hardik P atel and Dennis D avidson
9 C ytokine-Induced Neutrophil Chemotaxis Assay. . . . . . . . . . . . . . . . . . . . . . . 1 07
Dennis D avidson and Hardik P atel
10 A n In Vitro One-Dimensional Assay to Study Growth
Factor-Regulated Tumor Cell–Macrophage Interaction. . . . . . . . . . . . . . . . . . 115
Ved P. Sharma, B rian T. B eaty, D ianne C ox, J ohn S. C ondeelis,
and Robert J . Eddy
11 Chemotactic Responses by Macrophages to a Directional Source
of a Cytokine Delivered by a Micropipette. . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Michael Cammer and Dianne C ox
vii
viii Contents
12 Assessment of Phagocytic Activity of Cultured Macrophages
Using Fluorescence Microscopy and Flow Cytometry . . . . . . . . . . . . . . . . . . . 137
Lokesh S harma, Wenjun W u, S anjay L . D holakiya, Samir Gorasiya,
Jiao W u, R avikumar S itapara, V ivek Patel, Mao Wang, Michelle Zur,
Shloka R eddy, N athan S iegelaub, K atrina B amba, F rank A . Barile,
and Lin L . M antell
13 Assessment of Cytokine-Modulated Proteasome Activity. . . . . . . . . . . . . . . . . 1 47
Christopher J. K irk, S aul R. Powell, and E dmund J . Miller
14 E valuation of the Adverse Effect of Low Concentration of Cadmium
on Interleukin-4 Induced Class Switch Recombination
in Burkett’s Lymphoma Raji Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63
Vladimir Poltoratsky
15 A Mix-and-Measure Assay for Determining the Activation Status
of Endogenous Cdc42 in Cytokine-stimulated Macrophage Cell Lysates. . . . . 173
Veronika M iskolci, Désirée Spiering, D ianne Cox, and L ouis H odgson
16 A nalysis of the Cell Surface Expression of Cytokine Receptors
Using the Surface Protein Biotinylation Method. . . . . . . . . . . . . . . . . . . . . . . 185
Mahmud Arif Pavel, C larissa Lam, P arul Kashyap,
Zahra S alehi- Najafabadi, G urpreet S ingh, and Y ong Yu
17 Detection of CXCR2 Cytokine Receptor Surface Expression
Using Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Clarissa L am, M ahmud A rif P avel, Parul Kashyap,
Zahra Salehi-N ajafabadi, V ictoria V alentino, and Y ong Y u
18 Detecting Tie2, an Endothelial Growth Factor Receptor,
by Using Immunohistochemistry in Mouse Lungs. . . . . . . . . . . . . . . . . . . . . . 201
Prajna P . Guha, S ascha A . D avid, and Chandra C . Ghosh
19 Use of shRNA for Stable Suppression of Chemokine
Receptor Expression and Function in Human Cancer Cell Lines. . . . . . . . . . . 209
Nicole S alazar, D aniel M uñoz, J ames H oy, and Bal L. Lokeshwar
PART III ANALYSIS OF CYTOKINE INTRACELLULAR PROTEIN
LEVELS AND GENE EXPRESSION
20 Intracellular Staining and Detection of Cytokines
by Fluorescence-Activated Flow Cytometry. . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Giulia Freer
21 C ytokine Detection by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 35
Jian-Ge Qiu, X iao-Long Mei, Z he-Sheng Chen, and Zhi S hi
22 Analysis of IL-17 Production by Flow Cytometry and ELISPOT Assays . . . . . 2 43
Ling Zhao, Yuan Chou, Yanfang J iang, Zhenyu Jiang,
and C ong-Qiu Chu
23 Interleukin-1 (IL-1) Immunohistochemistry Assay in Oral Squamous
Cell Carcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Takaaki Kamatani
Contents ix
24 Immunofluorescence and Subsequent Confocal Microscopy of Intracellular
TNF in Human Neutrophils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Janet R ollins and Veronika Miskolci
25 Evaluating Cytoplasmic and Nuclear Levels of Inflammatory
Cytokines in Cancer Cells by Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . 271
Himavanth R . G atla, Bipradeb S ingha, Valerie P ersaud,
and Ivana Vancurova
26 W estern Analysis of Intracellular Interleukin-8 in Human
Mononuclear Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 85
Veronika Miskolci, L ouis H odgson, D ianne C ox, and I vana V ancurova
27 Quantitative Analysis of Bortezomib-Induced IL-8 Gene
Expression in Ovarian Cancer Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Bipradeb S ingha, S ai A . P hyo, H imavanth R . Gatla,
and Ivana V ancurova
28 Analysis of LPS-Induced, NFκB-Dependent Interleukin-8
Transcription in Kidney Embryonic Cell Line Expressing TLR4
Using Luciferase Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 05
Tamara Y unusova, M umtaz A khtar, and Vladimir Poltoratsky
29 C hromatin Immunoprecipitation Analysis of Bortezomib-M ediated
Inhibition of NFκB Recruitment to IL-1β and TNFα Gene
Promoters in Human Macrophages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Shannon S anacora, T zu-Pei C hang, and Ivana V ancurova
30 Analysis of TGFβ1 and IL-10 Transcriptional Regulation
in CTCL Cells by Chromatin Immunoprecipitation . . . . . . . . . . . . . . . . . . . . 329
Tzu-Pei C hang, M yra K im, and Ivana V ancurova
31 R adiolabeled Semi-quantitative RT-PCR Assay for the Analysis
of Alternative Splicing of Interleukin Genes. . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Felitsiya Shakola, S tephen B yrne, Kainaat J aved, and M atteo Ruggiu
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 63