Cargando…
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...
Autores principales: | , , , , , , , , , |
---|---|
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 |
_version_ | 1782120942217986048 |
---|---|
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. |
format | Text |
id | pubmed-200973 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2003 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT sunimariaa performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT dunnhollis performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT orrpatricial performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT laatriande performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT sinclairelizabeth performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT ghanekarsmitaa performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT bredtbarrym performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT dunnejohnf performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT mainovernonc performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis AT maeckerholdent performanceofplatebasedcytokineflowcytometrywithautomateddataanalysis |