TY  - JOUR
A1  - Karulin, Alexey Y.
A1  - Karacsony, Kinga
A1  - Zhang, Wenji
A1  - Targoni, Oleg S.
A1  - Moldova, Ioana
A1  - Dittrich, Marcus
A1  - Sundararaman, Srividya
A1  - Lehmann, Paul V.
T1  - ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting
JF  - Cells
N2  - 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.
KW  - ELISPOT
KW  - software
KW  - IFN-γ
KW  - IL-17
KW  - T cells
KW  - Normal Distribution
KW  - spot size
KW  - gating
KW  - cytokines
KW  - IL-2
KW  - IL-4
KW  - IL-5
KW  - CD8
KW  - CD4
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149648
VL  - 4
IS  - 1
ER  - 
TY  - JOUR
A1  - Duschl, Albert
A1  - Jahn, Ute
A1  - Bertling, Claudia
A1  - Sebald, Walter
T1  - A comparison of assays for the response of primary human T-cells upon stimulation with interleukin-2, interleukin-4 and interleukin-7
N2  - The most commonly used assay to quantitate the response of peripheral T~cells upon stimulation with growth factors is determination of incorporated (JH]TdR. We compared thls test to three other methods: 1. direct countlog of cells with a Coulter type counter as reference assay, 2. a colorimetric assay using the tetrazolium dye 3-[ 4,S-dimethylthiazol-l-yl]-2,5diphenyl tetrazolium (MTT), which is a cheap and increasingly popular non-radioactive method and 3. incorporation of the thymidine analog 5-bromo-2'-deoxyuridine detection with a monoclonal antibody on cytospins. Primary human PHA-blasts from >30 healthy individuals were stimulated with IL-2, IL-4 aod IL-7 and assayed with up to four different methods. We discuss the advantages and disadvantages of the assays used and tbe effects of differences between cell preparations. We observed no significant variations between individuals for the dose dependence, but the relative emctency of IL4 compared to IL-2 and IL-7 was variable. This was probably due to the slower response observed upon stimulation with this factor.
KW  - T-Lymphozyt
KW  - Interleukin 2
KW  - Interleukin 4
KW  - Interleukin 7
KW  - T-cells
KW  - proliferation assays
KW  - IL-2
KW  - IL-4
KW  - IL-7
Y1  - 1992
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86750
ER  -