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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...

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Autores principales: Suni, Maria A, Dunn, Holli S, Orr, Patricia L, Laat, Rian de, Sinclair, Elizabeth, Ghanekar, Smita A, Bredt, Barry M, Dunne, John F, Maino, Vernon C, Maecker, Holden T
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2003
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC200973/
https://www.ncbi.nlm.nih.gov/pubmed/12952557
http://dx.doi.org/10.1186/1471-2172-4-9
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author Suni, Maria A
Dunn, Holli S
Orr, Patricia L
Laat, Rian de
Sinclair, Elizabeth
Ghanekar, Smita A
Bredt, Barry M
Dunne, John F
Maino, Vernon C
Maecker, Holden T
author_facet Suni, Maria A
Dunn, Holli S
Orr, Patricia L
Laat, Rian de
Sinclair, Elizabeth
Ghanekar, Smita A
Bredt, Barry M
Dunne, John F
Maino, Vernon C
Maecker, Holden T
author_sort Suni, Maria A
collection PubMed
description 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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spelling pubmed-2009732003-09-30 Performance of plate-based cytokine flow cytometry with automated data analysis Suni, Maria A Dunn, Holli S Orr, Patricia L Laat, Rian de Sinclair, Elizabeth Ghanekar, Smita A Bredt, Barry M Dunne, John F Maino, Vernon C Maecker, Holden T BMC Immunol Methodology Article 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. BioMed Central 2003-09-02 /pmc/articles/PMC200973/ /pubmed/12952557 http://dx.doi.org/10.1186/1471-2172-4-9 Text en Copyright © 2003 Suni et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
spellingShingle Methodology Article
Suni, Maria A
Dunn, Holli S
Orr, Patricia L
Laat, Rian de
Sinclair, Elizabeth
Ghanekar, Smita A
Bredt, Barry M
Dunne, John F
Maino, Vernon C
Maecker, Holden T
Performance of plate-based cytokine flow cytometry with automated data analysis
title Performance of plate-based cytokine flow cytometry with automated data analysis
title_full Performance of plate-based cytokine flow cytometry with automated data analysis
title_fullStr Performance of plate-based cytokine flow cytometry with automated data analysis
title_full_unstemmed Performance of plate-based cytokine flow cytometry with automated data analysis
title_short Performance of plate-based cytokine flow cytometry with automated data analysis
title_sort performance of plate-based cytokine flow cytometry with automated data analysis
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC200973/
https://www.ncbi.nlm.nih.gov/pubmed/12952557
http://dx.doi.org/10.1186/1471-2172-4-9
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