Kaluza software version 3.1v

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.

Anti-coagulated blood (EDTA 2 mM) obtained from immunized and sham immunized mice groups 24 h post challenge were used for CD4+ and CD8+ T-cell estimation. The blood samples were pooled and erythrocytes 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, washed twice with Dulbecco’s PBS and 10⁶ cells were stained with rat-anti-mouse FITC-labeled anti-CD3, APC-labeled anti-CD4, and PE-labeled anti-CD8 monoclonal antibodies (BD Biosciences). A minimum of 20,000 events were counted for each analysis using BD Accuri^TM C6 Flow cytometer (Becton-Dickinson) and results were analyzed using Kaluza software version 3.1v (Beckman Coulter, USA). The percent activated CD3+ T cells were electronic gated among total lymphocytes obtained and CD4+ and CD8+ of T cells population in the gated CD3+ T cells was determined (Williamson et al., 2005 (link)).