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Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systema...

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Autores principales: Kuerten, Stefanie, Batoulis, Helena, Recks, Mascha S., Karacsony, Edith, Zhang, Wenji, Subbramanian, Ramu A., Lehmann, Paul V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901103/
https://www.ncbi.nlm.nih.gov/pubmed/24710483
http://dx.doi.org/10.3390/cells1030409
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author Kuerten, Stefanie
Batoulis, Helena
Recks, Mascha S.
Karacsony, Edith
Zhang, Wenji
Subbramanian, Ramu A.
Lehmann, Paul V.
author_facet Kuerten, Stefanie
Batoulis, Helena
Recks, Mascha S.
Karacsony, Edith
Zhang, Wenji
Subbramanian, Ramu A.
Lehmann, Paul V.
author_sort Kuerten, Stefanie
collection PubMed
description T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work.
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spelling pubmed-39011032014-04-07 Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays Kuerten, Stefanie Batoulis, Helena Recks, Mascha S. Karacsony, Edith Zhang, Wenji Subbramanian, Ramu A. Lehmann, Paul V. Cells Article T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work. MDPI 2012-07-30 /pmc/articles/PMC3901103/ /pubmed/24710483 http://dx.doi.org/10.3390/cells1030409 Text en © 2012 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Kuerten, Stefanie
Batoulis, Helena
Recks, Mascha S.
Karacsony, Edith
Zhang, Wenji
Subbramanian, Ramu A.
Lehmann, Paul V.
Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title_full Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title_fullStr Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title_full_unstemmed Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title_short Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays
title_sort resting of cryopreserved pbmc does not generally benefit the performance of antigen-specific t cell elispot assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901103/
https://www.ncbi.nlm.nih.gov/pubmed/24710483
http://dx.doi.org/10.3390/cells1030409
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