Cells | |
ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting | |
Alexey Y. Karulin2  Kinga Karacsony2  Wenji Zhang2  Oleg S. Targoni2  Ioana Moldovan2  Marcus Dittrich1  Srividya Sundararaman2  Paul V. Lehmann2  | |
[1] M.D., Ph.D., Biocenter, University of Wuerzburg, Am Hubland, 97074 Würzburg, Germany; E-Mail:;Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA; E-Mails: | |
关键词: ELISPOT; Normal Distribution; software; spot size; gating; cytokines; IFN-γ; IL-2; IL-4; IL-5; IL-17; T cells; CD4; CD8; | |
DOI : 10.3390/cells4010056 | |
来源: mdpi | |
【 摘 要 】
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.
【 授权许可】
CC BY
© 2015 by the authors; licensee MDPI, Basel, Switzerland.
【 预 览 】
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RO202003190017151ZK.pdf | 1699KB | download |