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

Cells

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.