Pe anti mouse cd8 antibody

Flow Cytometry analysis to evaluate CD4+ and CD8+ T cell counts in immunized mice were performed as previously described^{59 (link)}. Briefly, anti-coagulant treated (EDTA 2 mM) peripheral blood samples obtained from r-PB and sham immunized mice (n = 3 each group) were used for estimation of CD4+ and CD8+ T cell counts. The erythrocytes were lysed with 1:10 diluted lysis solution (FACS lysing solution, BD) for 30 min in dark at 37 °C. After 30 min incubation cells were harvested and washed with Dulbecco’s PBS and 10⁶ cells were stained with FITC anti-mouse CD4+ antibodies (BioLegend, India) and PE anti-mouse CD8+ antibodies (BioLegend, India). A minimum of 20,000 events were counted for each analysis using BD FACS Verse Flow Cytometer (Becton-Dickson, Singapore) and results were analysed using Kaluza software version 3.1 v (Beckman Coulter, USA). The percent activated CD3+ cells were electronic gated among the total lymphocytes obtained and the CD4+ and CD8+ T cell populations in the gated CD3+ T cells were determined.

To evaluate the CD4+ and CD8+ T cell counts in immunized mice, Flow Cytometry analysis was performed according to Williamson et al. (27 (link)). Briefly, peripheral blood samples were collected from r-PAbxpB and sham immunized mice (n = 3 each group) on the 45^th day of the immunization schedule and treated with 2 mM EDTA as an anti-coagulant. The erythrocytes were lysed using 1:10 diluted lysis solution (FACS lysing solution, BD) incubated at 37°C for 30 min in dark. Post incubation the cells were washed twice with Dulbecco's PBS and 10⁶ cells were stained with FITC anti-mouse CD4+ antibodies (BioLegend, India) and PE anti-mouse CD8+ antibodies (BioLegend, India). A minimum of 20,000 events were counted for each analysis using BD FACS Verse Flow Cytometer (Becton-Dickson, Singapore) and results were analyzed using Kaluza software version 3.1v (Beckman Coulter, USA). The percent activated CD4+ and CD8+ T cell populations were determined on scatter plot.