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ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific bac...

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Autores principales: Karulin, Alexey Y., Karacsony, Kinga, Zhang, Wenji, Targoni, Oleg S., Moldovan, Ioana, Dittrich, Marcus, Sundararaman, Srividya, Lehmann, Paul V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381209/
https://www.ncbi.nlm.nih.gov/pubmed/25612115
http://dx.doi.org/10.3390/cells4010056
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author Karulin, Alexey Y.
Karacsony, Kinga
Zhang, Wenji
Targoni, Oleg S.
Moldovan, Ioana
Dittrich, Marcus
Sundararaman, Srividya
Lehmann, Paul V.
author_facet Karulin, Alexey Y.
Karacsony, Kinga
Zhang, Wenji
Targoni, Oleg S.
Moldovan, Ioana
Dittrich, Marcus
Sundararaman, Srividya
Lehmann, Paul V.
author_sort Karulin, Alexey Y.
collection PubMed
description Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
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spelling pubmed-43812092015-05-18 ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting Karulin, Alexey Y. Karacsony, Kinga Zhang, Wenji Targoni, Oleg S. Moldovan, Ioana Dittrich, Marcus Sundararaman, Srividya Lehmann, Paul V. Cells Article Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs. MDPI 2015-01-20 /pmc/articles/PMC4381209/ /pubmed/25612115 http://dx.doi.org/10.3390/cells4010056 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
Karulin, Alexey Y.
Karacsony, Kinga
Zhang, Wenji
Targoni, Oleg S.
Moldovan, Ioana
Dittrich, Marcus
Sundararaman, Srividya
Lehmann, Paul V.
ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title_full ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title_fullStr ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title_full_unstemmed ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title_short ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
title_sort elispots produced by cd8 and cd4 cells follow log normal size distribution permitting objective counting
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381209/
https://www.ncbi.nlm.nih.gov/pubmed/25612115
http://dx.doi.org/10.3390/cells4010056
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