%0 Journal Article
%T ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
%A Alexey Y. Karulin
%A Kinga Karacsony
%A Wenji Zhang
%A Oleg S. Targoni
%A Ioana Moldovan
%A Marcus Dittrich
%A Srividya Sundararaman
%A Paul V. Lehmann
%J Cells
%P 56-70
%D 2015
%I MDPI AG
%R 10.3390/cells4010056
%X 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-¦Ã, 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.
%K ELISPOT
%K Normal Distribution
%K software
%K spot size
%K gating
%K cytokines
%K IFN-¦Ã
%K IL-2
%K IL-4
%K IL-5
%K IL-17
%K T cells
%K CD4
%K CD8
%U http://www.mdpi.com/2073-4409/4/1/56