Cd127 clone afr34

The spleens and kidneys were harvested on the indicated days postinfection (p.i.). The kidneys were minced and digested with collagenase (100 U/ml in DMEM with 2% FBS, 200 U/ml penicillin, 200 µg/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, San Jose, CA). The cells were isolated by centrifugation on a 44% and 66% Percoll gradient. The spleens were passed through a 70-µm nylon cell strainer and then treated with ACK buffer (0.15 M NH₄Cl, 1 mM KHCO₃, 1 mM Na₂EDTA [pH 7.0]) to lyse red blood cells (RBCs). The cells were exposed to fixable viability dye eFluor 780 (eBioscience, San Diego, CA) and then surface stained in fluorescence-activated cell sorting (FACS) buffer (PBS [pH 7.2] with 1% bovine serum albumin [BSA] and 0.1% sodium azide) for 30 min at 4°C with H-2D^b LT359 tetramers (provided by the NIH Tetramer Core Facility, Atlanta, GA) and monoclonal antibodies (MAbs) to CD19, NK1.1, CD3, CD8α (clone 53-6.7; BioLegend, San Diego, CA), CD44 (clone IM7; eBioscience), KLRG1 (clone 2F1; BD Biosciences), and CD127 (clone AFR34; BioLegend). The samples were collected on a BD LSRFortessa or BD FACSCanto10 flow cytometer (San Jose, CA). Fluorescence-minus-one (FMO) samples were used to set positive gates for each surface molecule examined.

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.