Performance of Plate-based Cytokine Flow Cytometry with Automated Data Analysis

Overview

Journal BMC Immunol

Publisher Biomed Central

Specialty Allergy & Immunology

Date 2003 Sep 4

PMID 12952557

Citations 21

Authors

Maria A Suni

Holli S Dunn

Patricia L Orr

Rian de Laat

Elizabeth Sinclair

Smita A Ghanekar

Barry M Bredt

John F Dunne

Vernon C Maino

Holden T Maecker

Affiliations

Soon will be listed here.

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.

Conclusion: 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.

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