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

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. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis.When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.Cytokine flow cytometry (CFC) assays use short-term in vitro stimulation and intracellular cytokine staining to quantitate potentially rare populations of antigen-specific T cells (see [1,2] for reviews). Because of the multi-parametric readout of flow cytometry, CFC is an attractive alternative to ELISPOT assays, allowing the dissection of responses by various phenotypes of T cells [3,4]. However, ELISPOT assays have traditionally been more amenable to high-throughput analysis due to their plate-based nature and the availability of automated instrumentation to analyze ELISPOT plates.In order to provide similar advantages to the CFC platform, we optimized the ability to stimulate, process, and acquire CFC samples in 96-well or 24-well plates, using stimulation with the superantigen Staphylococcal enterotoxin B (SEB), as well as cytomegalovirus (CMV) or HIV antigens. The result was two protocols optimized for stimulation and processing of whole blood samples (in deep-well 24- or 96-well plates), and one protocol optimized