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. 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.


CONCLUSIONS
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.
Suni
Maria A
MA
BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA. maria_suni@bd.com
Dunn
Holli S
HS
Orr
Patricia L
PL
de Laat
Rian
R
Sinclair
Elizabeth
E
Ghanekar
Smita A
SA
Bredt
Barry M
BM
Dunne
John F
JF
Maino
Vernon C
VC
Maecker
Holden T
HT
D000465:Algorithms; D001331:Automation; D015925:Cryopreservation; D016207:Cytokines / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D006801:Humans; D012984:Software; D013601:T-Lymphocytes / Q000276:immunology; D013997:Time Factors
D016207:Cytokines; D005456:Fluorescent Dyes