Flow cytometry for Immunophenotyping
For analysis of surface markers, fresh PBMCs were incubated for 30 minutes at room temperature in Stain Buffer (BD Biosciences, San Jose, CA) with optimal concentrations of the fluorochrome conjugated antibodies. Lymphocyte subsets were determined using the monoclonal antibodies as follows- CD3 APCH7 (Clone - SK-7), CD4 BV480 (Clone - SK-3), CD8 FITC (Clone - RPA-T8), CD62L APC (Clone - Dreg 56), CCR7 PE (Clone - 2-L1-A), CD45RA BV421 (Clone - 5H9), CD56 BV480 (Clone - NCAM 16.2) and CD16 FITC (Clone - 3G8) (BD Biosciences, San Jose, CA). The cells were further washed with stain buffer, fixed with 1% Formaldehyde in PBS and acquired to obtain 100000 gated lymphocyte events on FACS Jazz (BD Biosciences, San Jose, CA). The data was analysed using FACS Flow Jo software V10.7 (BD Biosciences, San Jose, CA).