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ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting

Please always quote using this URN: urn:nbn:de:bvb:20-opus-149648
  • 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 cellEach 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.show moreshow less

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Metadaten
Author: Alexey Y. Karulin, Kinga Karacsony, Wenji Zhang, Oleg S. Targoni, Ioana Moldova, Marcus Dittrich, Srividya Sundararaman, Paul V. Lehmann
URN:urn:nbn:de:bvb:20-opus-149648
Document Type:Journal article
Faculties:Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):Cells
Year of Completion:2015
Volume:4
Issue:1
Pagenumber:56-70
Source:Cells 2015, 4(1), 56-70. DOI: 10.3390/cells4010056
DOI:https://doi.org/10.3390/cells4010056
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=PMC4381209
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Tag:CD4; CD8; ELISPOT; IFN-γ; IL-17; IL-2; IL-4; IL-5; Normal Distribution; T cells; cytokines; gating; software; spot size
Release Date:2019/01/22
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International