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ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells

Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor i...

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Autores principales: Hanson, Jodi, Sundararaman, Srividya, Caspell, Richard, Karacsony, Edith, Karulin, Alexey Y., Lehmann, Paul V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381210/
https://www.ncbi.nlm.nih.gov/pubmed/25643292
http://dx.doi.org/10.3390/cells4010071
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author Hanson, Jodi
Sundararaman, Srividya
Caspell, Richard
Karacsony, Edith
Karulin, Alexey Y.
Lehmann, Paul V.
author_facet Hanson, Jodi
Sundararaman, Srividya
Caspell, Richard
Karacsony, Edith
Karulin, Alexey Y.
Lehmann, Paul V.
author_sort Hanson, Jodi
collection PubMed
description Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.
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spelling pubmed-43812102015-05-18 ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells Hanson, Jodi Sundararaman, Srividya Caspell, Richard Karacsony, Edith Karulin, Alexey Y. Lehmann, Paul V. Cells Article Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format. MDPI 2015-01-29 /pmc/articles/PMC4381210/ /pubmed/25643292 http://dx.doi.org/10.3390/cells4010071 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hanson, Jodi
Sundararaman, Srividya
Caspell, Richard
Karacsony, Edith
Karulin, Alexey Y.
Lehmann, Paul V.
ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title_full ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title_fullStr ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title_full_unstemmed ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title_short ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
title_sort elispot assays in 384-well format: up to 30 data points with one million cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381210/
https://www.ncbi.nlm.nih.gov/pubmed/25643292
http://dx.doi.org/10.3390/cells4010071
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