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Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells
L E Nomura1, J M Walker, H T Maecker
1Biological Research and Development, BD Biosciences, Immunocytometry Systems, San Jose, CA 95131, USA.
Insights
Optimized flow cytometry methods improve cytokine analysis in CD4(+) T cells. This enhances the study of immune responses to pathogens and vaccines, offering greater convenience and reproducibility.
Area of Science:
- Immunology
- Cellular Biology
- Flow Cytometry
Background:
- Cytokine production analysis is crucial for understanding immune responses.
- Three-color flow cytometry is a sensitive method for evaluating cytokine production in individual CD4(+) T cells.
Purpose of the Study:
- To present optimized methods for acquiring and analyzing cytokine production.
- To enhance the detection of interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4).
Main Methods:
- Utilized cytomegalovirus (CMV) as an antigenic model in whole blood from CMV-seropositive donors.
- Performed titration and kinetic experiments to determine optimal antigen dose and incubation time.
- Introduced a timed cooling device and fourth-color reagents for improved sample handling and background reduction.
Main Results:
- Optimal CMV stimulation achieved with 5 microg/ml for at least 6 hours, including an initial 2-hour Brefeldin A-free period.
- Timed cooling effectively preserved sample integrity without affecting cell response.
- Fourth-color reagents successfully eliminated false positives from monocytes and activated platelets.
Conclusions:
- Optimized methods enhance convenience and reproducibility for whole blood cytokine analysis.
- Intra-assay variability was reduced to less than 10%, and inter-assay variability to less than 25%.
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%).
