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Cytometry. 2000 May 1;40(1):60-8.

Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells.

Author information

1
Biological Research and Development, BD Biosciences, Immunocytometry Systems, San Jose, CA 95131, USA.

Abstract

BACKGROUND:

The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells.

METHODS:

We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors.

RESULTS:

CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining.

CONCLUSIONS:

These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%).

PMID:
10754518
[Indexed for MEDLINE]

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