1887

Chapter 11.15 : Flow Cytometry Whole-Blood Intracellular-Cytokine Assay Using Phorbol Myristate Acetate, Ionomycin, and Brefeldin A

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in
Zoomout

Flow Cytometry Whole-Blood Intracellular-Cytokine Assay Using Phorbol Myristate Acetate, Ionomycin, and Brefeldin A, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817435/9781555815271_Chap11_15-1.gif /docserver/preview/fulltext/10.1128/9781555817435/9781555815271_Chap11_15-2.gif

Abstract:

Cytokines are soluble proteins produced by T and B lymphocytes, natural killer cells, monocytes, macrophages, and granulocytes. Specifically, cytokines regulate growth, differentiation, and function of a wide variety of cells and mediate normal and pathological responses. Most cytokine bioassays examine cytokines at the cell population level, but they cannot provide information concerning the phenotype of cytokine-producing cells or mechanism of cytokine products. The flow cytometry method described here measures production of cytokines by T-cell subsets using whole blood. Heparinized blood is stimulated with the polyclonal activator phorbol myristate acetate plus ionomycin (induces intracelluar signal cascades for polyclonal leukocyte activation) and the protein transport inhibitor brefeldin A (BFA) (a fungal metabolite that interferes with vesicular transport from the rough endoplasmic reticulum to the Golgi complex) for 4 h at 37°C and 5% CO. Activated cells are stained with monoclonal antibodies for lymphocyte surface markers, followed by lysing of RBCs. WBCs are fixed and permeabilized simultaneously and stained with monoclonal antibodies to intracellular cytokines. Stained cells are analyzed by flow cytometry.

Citation: Garcia L. 2010. Flow Cytometry Whole-Blood Intracellular-Cytokine Assay Using Phorbol Myristate Acetate, Ionomycin, and Brefeldin A, p 238-241. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch11.15
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

References

/content/book/10.1128/9781555817435.chap11.15
1. Centers for Disease Control and Prevention. 1997. Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb. Mortal. Wkly. Rep. 46(RR-2):129.
2. Jung, T.,, U. Schauer,, C. Heusser,, C. Nermann,, and C. Rieger. 1993. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159:197207.
3. Maino, V.,, M. A. Suni,, and J. J. Ruitenberg. 1995. Rapid flow cytometric method for measuring lymphocyte subset activation. Cytometry 20:127133.
4. Picker, L. J.,, M. K. Singh,, Z. Zdraveski,, J. R. Treer,, S. L. Waldrop,, P. R. Bergstresser,, and V. C. Maino. 1995. Direct demonstration of cytokine heterogeneity among human memory/effector T cells by flow cytometry. Blood 86:14081419.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error