Main description:

Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes and associated with a wide variety of diseases, including inflammatory disorders as well as many types of cancer and leukemia. Knowledge about the quantitative and qualitative nature of cytokine production is critical in the understanding of normal and pathological processes. The cytokine detection in biological and clinical samples faces many challenges including their 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 will provide a comprehensive collection of classic and cutting-edge methodologies that are currently used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. The chapters are divided into four main categories. The first group 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 activity and include ELISA, flow cytometry, ELISPOT and the antibody-based proximity ligation. The second group will focus on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced cell degranulation assay, cell proliferation and differentiation, cytokine-induced cytokine production and the radioreceptor cytokine assay. The third group focuses on the analysis of intracellular cytokines by flow cytometry, western blotting and fluorescence and confocal microscopy. In addition, this category includes protocols for quantitative analysis of cytokine gene expression by real time RT-PCR and analysis of the cytokine promoter occupancy by chromatin immunoprecipitation. The fourth group focuses on the recently developed multiplex arrays that can measure multiple cytokines in the same sample at the same time. This group includes quantification of multiple cytokines using cytometric bead arrays, ELISPOT assays, proteomics cytokine evaluation, multiplexed proximity ligation assays for high-throughput cytokine analysis and finally, cytokine gene expression analysis by gene arrays. The protocols will be written by experienced basic and clinical researchers with hands-on knowledge of the described protocols. By covering a broad variety of methods used in cytokine detection and analysis, this book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.

Contents:

Part I: Detection of Released Cytokines by Immunoassays

The Challenge of Measuring Elusive Immune Markers by Enzyme Linked Immuno-Spot (ELISPOT) Technique

Maria Faresjoe

Analysis of the Released Nuclear Cytokine HMGB1 in Human Serum

Haichao Wang, Lin Zhao, Jianhua Li, Shu Zhu, and Meggie Yeung

ELISA Based Assay for IP-10 Detection from Filter Paper Samples

Camilla Heldbjerg Drabe, Thomas Blauenfeldt, and Morten Ruhwald

Enhanced ELISA Based on Carboxymethylated Dextran Coatings

Benoit Liberelle, Charles Fortier, and Gregory De Crescenzo

A Novel Three-Dimensional Biosensor Based on Aluminum Oxide: Application for Early Stage Detection of Human Interleukin-10

Michael Lee, Abdoullatif Baraket, Nadia Zine, Miguel Zabala, Francesca Campabadal, Raffaele Caruso, Maria Giovanna Trivella, Nicole Jaffrezic-Renault, and Abdelhamid Errachid

6. Quantification of Multiple Cytokines and Chemokines Using Cytometric Bead Arrays

Gemma Moncunill, Joseph J. Campo, and Carlota Dobano

7. A Useful Guide for Analysis of Immune Markers by Fluorochrome (Luminex) Technique

Maria Faresjoe

Part II: Cytokine Bioassays

8. Control of Pro-inflammatory Cytokine Release from Human Monocytes with the Use of an Interleukin-10 Monoclonal Antibody

Hardik Patel and Dennis Davidson

9. Cytokine-Induced Neutrophil Chemotaxis Assay

Dennis Davidson and Hardik Patel

10. An In Vitro One-Dimensional Assay to Study Growth Factor-Regulated Tumor Cell-Macrophage Interaction

Ved P. Sharma, Brian T. Beaty, Dianne Cox, John S. Condeelis, and Robert J. Eddy

11. Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette

Michael Cammer and Dianne Cox

12. Assessment of Phagocytic Activity of Cultured Macrophages Using Fluorescence Microscopy and Flow Cytometry

Lokesh Sharma, Wenjun Wu, Sanjay L. Dholakiya, Samir Gorasiya, Jiao Wu, Ravikumar Sitapara, Vivek Patel, Mao Wang, Michelle Zur, Shloka Reddy, Nathan Siegelaub, Katrina Bamba, Frank A. Barile, and Lin L. Mantell

13. Assessment of Cytokine-Modulated Proteasome Activity

Christopher J. Kirk, Saul R. Powell, and Edmund J. Miller

14. Evaluation of the Adverse Effect of Low Concentration of Cadmium on Interleukin-4 Induced Class Switch Recombination in Burkett's Lymphoma Raji Cell Line

Vladimir Poltoratsky

15. A Mix-and-Measure Assay for Determining the Activation Status of Endogenous Cdc42 in Cytokine-Stimulated Macrophage Cell Lysates

Veronika Miskolci, Desiree Spiering, Dianne Cox, and Louis Hodgson

16. Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method

Mahmud Arif Pavel, Clarissa Lam, Parul Kashyap, Zahra Salehi-Najafabadi, Gurpreet Singh, and Yong Yu

17. Detection of CXCR2 Cytokine Receptor Surface Expression Using Immunofluorescence

Clarissa Lam, Mahmud Arif Pavel, Parul Kashyap, Zahra Salehi-Najafabadi, Victoria Valentino, and Yong Yu

18. Detecting Tie2, an Endothelial Growth Factor Receptor, by Using Immunohistochemistry in Mouse Lungs

Prajna P. Guha, Sascha A. David, and Chandra C. Ghosh

19. Use of shRNA to Stably Suppress Chemokine Receptor Expression and Function in Human Cancer Cell Lines

Nicole Salazar, Daniel Munoz, James Hoy, 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 Giulia Freer

21. Cytokine Detection by Flow Cytometry

Jian-Ge Qiu, Xiao-Long Mei, Zhe-Sheng Chen, and Zhi Shi

22. Analysis of IL-17 Production by Flow Cytometry and ELISPOT Assays

Ling Zhao, Yuan Chou, Yanfang Jiang, Zhenyu Jiang, and Cong-Qiu Chu

Interleukin-1 (IL-1) Immunohistochemistry Assay in Oral Squamous Cell Carcinoma

Takaaki Kamatani

24. Immunofluorescence Confocal Microscopy of Intracellular TNF in Human Neutrophils

Janet Rollins and Veronika Miskolci

25. Evaluating Cytoplasmic and Nuclear Levels of Inflammatory Cytokines in Cancer Cells by Western Blotting

Himavanth R. Gatla, Bipradeb Singha, Valerie Persaud, and Ivana Vancurova

26. Western Analysis of Intracellular Interleukin-8 in Human Mononuclear Leukocytes

Veronika Miskolci, Louis Hodgson, Dianne Cox, and Ivana Vancurova

27. Quantitative Analysis of Bortezomib Induced IL-8 Gene Expression in Ovarian Cancer Cells Bipradeb Singha, Sai A. Phyo, Himavanth R. Gatla, and Ivana Vancurova

28. Analysis of LPS-Induced, NFkB-Dependent Interleukin-8 Transcription in Kidney Embryonic Cell Line Expressing TLR4 Using Luciferase Assay

Tamara Yunusova, Mumtaz Akhtar, and Vladimir Poltoratsky

29. Chromatin Immunoprecipitation Analysis of Bortezomib Mediated Inhibition of NF B Recruitment to IL-1b and TNFa Gene Promoters in Human Macrophages

Shannon Sanacora, Tzu-Pei Chang, and Ivana Vancurova

30. Analysis of TGFb1 and IL-10 Transcriptional Regulation in CTCL Cells by Chromatin Immunoprecipitation

Tzu-Pei Chang, Myra Kim, and Ivana Vancurova

31. Radiolabeled Semi-Quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes

Felitsiya Shakola, Stephen Byrne, Kainaat Javed, and Matteo Ruggiu