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.