Cd62l clone mel 14

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.

All antibodies were purchased from BioLegend except where otherwise noted. Following isolation, cells were stained with fluorochrome-conjugated antibodies against mouse CD3 (clone 17A2), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD44 (clone 1M7), CD62L (clone Mel-14), and T cell receptor Vβ8.3 (clone 1B3.3) (BD Biosciences) along with anti-FcRγ (Bio X Cell, West Lebanon, NH) and a LIVE/DEAD Fixable Aqua Dead cell stain kit to exclude dead cells (Invitrogen). To assess T-bet⁺ expression, a subset of skewed cells were fixed and permeabilized using an Invitrogen transcription factor fixation/permeabilization kit (Invitrogen) and staining with an anti-T-bet antibody (clone 4B10). AccuCheck counting beads (Invitrogen) were used to determine absolute cell counts. Data were collected on an LSR II flow cytometer (BD Biosciences) and were analyzed using FlowJo (Tree Star, Ashland, OR).

For surface staining, splenocytes and isolated immune cells from spleen, muscle, and lymph node tissues were stained for 15 min at room temperature with antibodies against the following proteins: CD4 (clone GK1.5, 863 eBioscience; clone H129.19, BioLegend, CA, USA), CD8 (clone 53-6.7, BD Biosciences; clone 53-6.7, Invitrogen, CA, USA), CD44 (clone IM7, Invitrogen), CD62L (clone MEL 14, BD Biosciences), CD11b (clone M1/70, Bio Legend), F4/80 (clone BM8, Invitrogen), CD86 (clone GL1, BD Biosciences), and CD11c (clone N48, eBioscience). Cells were fixed with 1% paraformaldehyde, analyzed using a FACS Canto II flow cytometer (BD Biosciences, NJ, USA), and the data were analyzed using FlowJo software (TreeStar, OR, USA).

Flow cytometry was performed on the Attune NxT Flow Cytometer (Life Technologies, CA, USA). Cells were first gated for doublet exclusion [forward scatter height (FSC-H) vs. forward scatter area (FSC-A)] followed by side scatter height (SSC-H) vs. FSC-H gating. Cell viability was checked by negative gating of cells stained with “Live/Dead Fixable Aqua Dead Cell Staining Kit” (Life Technologies, CA, USA). Neutrophils were analyzed by detecting the surface antigens with the following antibodies: Ly6G (clone 1A-8, BD Biosciences, CA, USA), CD11b (clone M1/70, Biolegend, CA, USA), CD54 (clone 3E2), and CD62L (clone MEL-14) from BD Pharmingen, CA, USA; or total antigens (surface and intracellular) by antibodies: CXCR4 (clone L276F12) and CXCR2 (clone SA045E1) from Biolegend, CA, USA; or by intracellular labeling with antibodies: Arg-1 (clone A1exF5) and inducible nitric oxide synthase (iNOS) (clone CXNFT) from Invitrogen, MA, USA. Cells were fixed with 1% paraformaldehyde and permeabilized with Saponin buffer [0.1% Saponin (w/v), 0.1% bovine serum albumin (BSA), 0.01 M HEPES, and 0.1% sodium azide in phosphate-buffered saline (PBS)] prior to the intracellular labeling as described previously (18 (link)).