ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting
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- 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.…
Autor(en): | Alexey Y. Karulin, Kinga Karacsony, Wenji Zhang, Oleg S. Targoni, Ioana Moldova, Marcus Dittrich, Srividya Sundararaman, Paul V. Lehmann |
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URN: | urn:nbn:de:bvb:20-opus-149648 |
Dokumentart: | Artikel / Aufsatz in einer Zeitschrift |
Institute der Universität: | Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften |
Sprache der Veröffentlichung: | Englisch |
Titel des übergeordneten Werkes / der Zeitschrift (Englisch): | Cells |
Erscheinungsjahr: | 2015 |
Band / Jahrgang: | 4 |
Heft / Ausgabe: | 1 |
Seitenangabe: | 56-70 |
Originalveröffentlichung / Quelle: | Cells 2015, 4(1), 56-70. DOI: 10.3390/cells4010056 |
DOI: | https://doi.org/10.3390/cells4010056 |
PubMed-ID: | https://pubmed.ncbi.nlm.nih.gov/PMC4381209 |
Allgemeine fachliche Zuordnung (DDC-Klassifikation): | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Freie Schlagwort(e): | CD4; CD8; ELISPOT; IFN-γ; IL-17; IL-2; IL-4; IL-5; Normal Distribution; T cells; cytokines; gating; software; spot size |
Datum der Freischaltung: | 22.01.2019 |
Lizenz (Deutsch): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |