Performance of plate-based cytokine flow cytometry with automated data analysis.

Background: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates.

Results: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates.

Dynamics of CD4 and CD8 T cell responses to cytomegalovirus in healthy human donors.

To study the dynamics of cytomegalovirus (CMV) immunity in healthy immunocompetent hosts, interferon-gamma-producing CD4 and CD8 T cell responses in the presence or absence of CMV antigens were measured from 15 CMV-seropositive donors and 13 CMV-seronegative donors. Cytokine responses in the absence of antigen were significantly higher in CMV-seropositive donors. Also, a disproportionate number of CD69(+) cells isolated ex vivo from CMV-seropositive donors were specific for CMV, suggesting recent reactivation in vivo.