TY  - JOUR
A1  - Karulin, Alexey Y.
A1  - Caspell, Richard
A1  - Dittrich, Marcus
A1  - Lehmann, Paul V.
T1  - Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses
JF  - Cells
N2  - Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 – 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.
KW  - ELISPOT
KW  - statistics
KW  - t-Test
KW  - ANOVA
KW  - T-cells
KW  - normal distribution
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149968
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  -