Anti cd62l mel 14

Single-cell suspensions were prepared from the thymus and spleens of Paxbp1 cKO and control mice. For cell surface marker staining, 2 million cells were incubated with the fluorescent antibody mixture at 4°C for 30 min, washed with PBS, and then analyzed by FACS. For intracellular staining, cells were stained with appropriate surface markers, then fixed and stained with antibodies according to the manufacturer’s protocol for the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend, 428703). Zombie Violet™ Fixable Viability Kit (Biolegend, 423113) or 7-AAD (BD, 420404) was used to mark dead cells. The Backman Coulter CytoFLEX S (Backman) was used for the flow cytometric analysis. For cell sorting, thymocytes were isolated and stained with surface markers as indicated above. Then labeled cells were resuspended in FACS buffer (1% FBS + 2 mM EDTA, 25 mM HEPES in PBS). DN, ISP, DP, and SP thymocytes were sorted on an FACSAria III (BD Biosciences).
The following antibodies were used: anti-CD4 (RM-5), anti-CD8 (53–6.7), anti-TCRβ (H57–597), anti-CD44 (IM7), anti-CD25 (PC61), anti-TCRγ/δ (GL3), anti-CD5 (53–7.3), anti-CD69 (H1.2F3), and anti-CD19 (6D5), all of which were purchased from BioLegend. Anti-CD62L (MEL-14) was purchased from Invitrogen.

Expression of activation‐associated surface molecules on OT‐I CTLs was evaluated on day 3 or 4 post‐transduction or the day that adoptive transfer was performed, which was usually day 6 post‐isolation. Cells were stained with anti‐CD8α (53–6.7, BD Biosciences, San Jose, CA, USA), anti‐CD44 (IM7, BD Biosciences), anti‐CD25 (PC61, BD Biosciences), anti‐CD69 (H1.2F3, BD Biosciences), anti‐CD62L (MEL‐14, eBioscience, Vienna, Austria) and anti‐V_α2 (B20.1, BD Biosciences). Antibodies were used at 1 μg mL⁻¹ in running buffer (5% fetal bovine serum and 2 mm ethylenediaminetetraacetic acid, 0.01% sodium azide in 1 × phosphate‐buffered saline) and the cells were stained for 20 min at 4°C prior to washing two times with running buffer. Cell viability was evaluated using 0.5 μg mL⁻¹ 4,6‐diamidino‐2‐phenylindole (Thermo Fisher Scientific) or the LIVE/DEAD Fixable near‐IR (Thermo Fisher Scientific). Data were collected on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For fluorescence‐activated cell sorting of medium to high mCherry expressing CTL population, cells were resuspended in FACS buffer (5% fetal calf serum, 2 mm ethylenediaminetetraacetic acid in 1 × phosphate‐buffered saline) and incubated with 4,6‐diamidino‐2‐phenylindole. Cells were either used fresh or cryopreserved according to established methods.85