Effects of Different Media on Human T Regulatory Cells Phenotype

MAGDALENA GŁACZYNSKA; MAJA MACHCINSKA; KATARZYNA DONSKOW-LYSONIEWSKA

doi:10.21873/invivo.12257

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Figure 1.
Viability of PBMC incubated overnight at 4°C in PBS, PBS containing 0.5% BSA, RPMI 1640 and RPMI 1640 containing 10% FBS, determined by flow cytometry. Gating strategy for flow cytometric analysis of the leukocyte (granulocytes and lymphocytes) and lymphocyte population to identify live/dead cells. A time gate was initially applied to exclude any electronic noise and artifact (not shown here). Next, based on size and granularity, lymphocytes and leukocytes were gated in a forward scatter area (FSC-A) versus side scatter area (SSC-A) plot (A). Then, separately for leukocyte (B) and lymphocyte (C) populations, doublet cells were excluded using FSC-A/FSC-height (FSC-H), FSC-A/FSC-Width and SSC-A/SSC-height (FSC-H) parameters. Within the singlet cell population, viable leukocytes (D) and lymphocytes (E) were gated based on the dim expression of the Fixable Viability Dye (FVD). Results are average percentages of viable cells for all donors (n=10). Representative plots are presented.
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Figure 2.
Viability of PBMCs incubated overnight at 4°C in PBS, PBS containing 0.5% BSA, RPMI 1640 and RPMI 1640 containing 10% FBS, as determined by the Muse Cell Analyzer. The representative plots show the average percentage of live and dead PBMCs stored overnight at 4°C in various media: A) PBS; B) PBS containing 0.5% BSA; C) RPMI 1640; D) RPMI 1640 containing 10% FBS.
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Figure 3.
Effect of overnight incubation at 4°C in different media on CD4⁺ and CD8⁺ Tregs. Freshly isolated and overnight incubated at 4°C in different media (PBS, PBS containing 0.5% BSA, RPMI 1640 and RPMI 1640 containing 10% FBS) CD4⁺ and CD8⁺ Tregs were analyzed by FACS. The gating strategy of CD4⁺ and CD8⁺ Tregs is presented on dot plots from one representative donor. Lymphocytes were identified based on their forward- and side-scatter properties, and doublet cells and dead cells were eliminated through the use of a viability dye (show in Figure 1), followed by expression of CD3 (A). CD8 Tregs were next identified by CD8 and CD25 (B) and FoxP3 (C) expression. CD4 Tregs were identified as CD4⁺CD127^low/- cells (E) with CD25 and FoxP3 co-expression (F). The graphs display the percentages (the mean percentage values±SD) of CD4⁺CD127^low/-CD25⁺FoxP3⁺ Tregs and CD8⁺CD25⁺FoxP3⁺ Tregs for all donors (n=10). *p<0.05 (ANOVA or Kruskal-Wallis).