Flow Cytometry Assay:
The sample preparation for flow cytometric analysis was done by stain-lyse-wash protocol. The antibodies utilized in the assay were procured from Becton Dickinson (BD) Biosciences, San Jose, California, USA. The fluorochrome and clones of antibody used were CD3 PerCP-Cy5.5 (SP34-2)/ CD4 PE-Cy7 (SK3)/ CD8 APC-H7 (SK1)/ CD197 APC (2-L1-A)/ CD95 BV421 (DX2)/ CD45RA BV480 (H1100)/ CD27 PE (M-T271) in the memory cell tube. A blank tube without antibody was also run. While FACS Canto II flow cytometer, BD Biosciences was used for sample acquisition, data analysis was performed using FACS Diva version 8 software. At least 10,000 CD3+T cells were acquired in each case to ensure adequate number of subset population to be studied. Based on light scatter properties, lymphocytes were identified. From this population, CD3+ total T-cells as well as CD4+ and CD8+ subsets were subsequently defined. On the basis of differential expression of CD45RA and CD197 (CCR7), central memory (CM), effector memory (EM) and effector memory RA (EMRA) subsets were identified on both helper and cytotoxic cell. From the CD45RA+CD197+ gate, CD95+ stem cell memory and CD95- naïve T-cells were further identified. The gating strategy has been depicted in figure 1. (Figure-1)
Statistical analysis:
Normality assessment of data was done with Shapiro Wilk test. Continuous data was expressed as mean (with standard deviation) or median (with range) with respect to its distribution. To compare the expression of antibodies, Mean fluorescence intensity (MFI) was used. Non-parametric assessments were used to compare the different groups; 2-independent sample analysis was done using Kolmogorov-Smirnov Z test while >2-independent sample analysis was done by Kruskal Wallis ANOVA with post-hoc pairwise comparisons. All assessments were 2-tailed and a P-value of <0.05 was taken as significant; all values <0.001 were expressed as <0.001. Data was analyzed with IBM SPSS package for Windows, Version 24.0, Armonk, NY.