title: Performance of plate-based cytokine flow cytometry with automated data analysis.
abstract: 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.
authors.affiliation: BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA. maria_suni@bd.com
authors.forename: Maria A
authors.initials: MA
authors.lastname: Suni
authors.forename: Holli S
authors.initials: HS
authors.lastname: Dunn
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Orr
authors.forename: Rian
authors.initials: R
authors.lastname: de Laat
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sinclair
authors.forename: Smita A
authors.initials: SA
authors.lastname: Ghanekar
authors.forename: Barry M
authors.initials: BM
authors.lastname: Bredt
authors.forename: John F
authors.initials: JF
authors.lastname: Dunne
authors.forename: Vernon C
authors.initials: VC
authors.lastname: Maino
authors.forename: Holden T
authors.initials: HT
authors.lastname: Maecker
mesh_terms: 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
chemical_list: D016207:Cytokines; D005456:Fluorescent Dyes