14964
2015
eng
56-70
1
4
article
1
2017-06-02
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ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting
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.
Cells
10.3390/cells4010056
PMC4381209
urn:nbn:de:bvb:20-opus-149648
Cells 2015, 4(1), 56-70. DOI: 10.3390/cells4010056
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Kinga Karacsony
Wenji Zhang
Oleg S. Targoni
Ioana Moldova
Marcus Dittrich
Srividya Sundararaman
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
software
eng
uncontrolled
IFN-γ
eng
uncontrolled
IL-17
eng
uncontrolled
T cells
eng
uncontrolled
Normal Distribution
eng
uncontrolled
spot size
eng
uncontrolled
gating
eng
uncontrolled
cytokines
eng
uncontrolled
IL-2
eng
uncontrolled
IL-4
eng
uncontrolled
IL-5
eng
uncontrolled
CD8
eng
uncontrolled
CD4
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14964/094_Karulin_Cells.pdf
14996
2015
eng
96-111
1
4
article
1
2017-06-08
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Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses
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.
Cells
10.3390/cells4010096
PMC4381212
urn:nbn:de:bvb:20-opus-149968
Cells 2015, 4(1), 96-111. DOI: 10.3390/cells4010096
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Richard Caspell
Marcus Dittrich
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
statistics
eng
uncontrolled
t-Test
eng
uncontrolled
ANOVA
eng
uncontrolled
T-cells
eng
uncontrolled
normal distribution
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14996/108_Karulin_Cells.pdf
15021
2015
eng
40-55
1
4
article
1
2017-06-09
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Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Cells
10.3390/cells4010040
urn:nbn:de:bvb:20-opus-150213
Cells 2015, 4:1, 40-55. DOI: 10.3390/cells4010040
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Marie Wunsch
Richard Caspell
Stefanie Kuerten
Paul V. Lehmann
Srividya Sundararaman
eng
uncontrolled
T cell assay
eng
uncontrolled
apoptosis
eng
uncontrolled
acceptance
eng
uncontrolled
viability
eng
uncontrolled
ELISPOT
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15021/129_Wunsch_Cells.pdf