TY - JOUR
T1 - Optimization of whole blood antigen-specific cytokine assays for CD4+ T cells
AU - Nomura, Laurel E.
AU - Walker, Joshua M.
AU - Maecker, Holden T.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 2000/5/1
Y1 - 2000/5/1
N2 - 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 (IFNγ), tumor necrosis factor alpha (TNFα), 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 μg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 μg/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°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%). (C) 2000 Wiley-Liss, Inc.
AB - 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 (IFNγ), tumor necrosis factor alpha (TNFα), 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 μg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 μg/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°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%). (C) 2000 Wiley-Liss, Inc.
KW - CMV
KW - Flow cytometry
KW - Intracenular cytokines
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U2 - 10.1002/(SICI)1097-0320(20000501)40:1<60::AID-CYTO8>3.0.CO;2-J
DO - 10.1002/(SICI)1097-0320(20000501)40:1<60::AID-CYTO8>3.0.CO;2-J
M3 - Article
C2 - 10754518
AN - SCOPUS:0034192241
VL - 40
SP - 60
EP - 68
JO - Communications in Clinical Cytometry
JF - Communications in Clinical Cytometry
SN - 0196-4763
IS - 1
ER -