Cd3e clone 145 2c11

Tissue sequestered leukocytes were isolated and processed as described earlier^{43 (link)}. Briefly, brain from uninfected and infected mice (7 d.p.i), were used for the purpose. Brains were harvested from different groups of animals after cardiac puncture and whole body perfusion. Brains were maintained at 4 ^◦C in the serum containing DMEM media until digestion. Each brain was cut into pieces and digested with 0.05% collagenase D (Roche) and 2U/ml of DNaseI (Thermo scientific) at 37 ^◦C for 20 min. The tissue extract was sieved through nylon mesh (70 μm) and centrifuged at 400xg for 5 min. The pellet was resuspended in DMEM medium and layered on 30% (v/v) percoll (Sigma Aldrich) and centrifuged at 400xg for 20 min. The cell suspension was treated with RBC lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.3) to remove RBCs. Cell viability was checked using Trypan blue (Sigma Aldrich) and 10⁷ cells were used for flow cytometry. Cell surface staining was performed using conjugated CD3e (clone145-2C11, BD Biosciences), CXCR3/CD183 (clone CXCR3-173, eBioscience), CD8a antibodies (clone53-6.7, BD Biosciences) for 1 h at 4 ^◦C. Cells were washed with DMEM and fixed with 4% paraformaldehyde followed by PBS wash. Cells were finally suspended in PBS and used for FACS analysis in BD FACS Canto™ II machine. Results were analyzed using BD FACS DIVA™ software.