Cd11a clone 2d7

Spleens and brains were harvested at indicated days p.i. Brains were minced and digested with collagenase (100 U/ml in DMEM with 2% FBS, 200 U/ml penicillin, 200 g/ml streptomycin, 2 mM L-glutamine, 5 μM HEPES, 1 μM MgCl₂, 1 μM CaCl₂) for 30 min at 37°C, passed through a 70 μm nylon cell strainer (BD Biosciences). Cells were isolated by centrifugation on a 44%:66% Percoll gradient. Spleens were passed through a 70 μm nylon cell strainer, then treated with ACK buffer (0.15 M NH₄Cl, 1mM KHCO₃, 1mM Na₂EDTA, pH 7.0) to lyse RBCs. Cells were surface-stained in FACS Buffer (PBS, pH 7.2 with 1% BSA, 0.1% sodium azide) for 30 min at 4°C with mAbs to CD8α (clone 53–6.7; Biolegend), CD44 (clone IM7; eBioscience), CD45.1 (clone A20; Biolegend), CD62L (clone MEL-14; BD Biosciences), CD69 (clone H1.2F3; BD Biosciences), PD-1 (clone RMP1-30; Biolegend), CD11a (clone 2D7; BD Biosciences), CD49d (clone MFR4.B; BioLegend), KLRG1 (clone 2F1; BD Biosciences), and CD127 (clone AFR34; Biolegend). Samples were collected on a BD LSR Fortessa or FACSCanto10 flow cytometer. Fluorescence-minus-one (FMO) samples were used to set positive gates for each surface molecule examined.