Anti cd44 pe cy7 clone im7

Mouse splenocytes were prepared as described above. Samples were resuspended in PBS with 1% fetal calf serum, followed by incubation with anti-CD16/32 mAb (Fc block, clone 93, BioLegend) for 10 min at 4 °C to prevent nonspecific Fc receptor binding. Samples were then stained for 40 min at 4 °C with various combinations of fluorochrome-conjugated antibodies: anti-CD45-PerCP (clone 30-F11, BioLegend), anti-CD8α-APC (clone 53–6.7, BD Biosciences), anti-CD3-Alexa Fluor 488 (clone 145-2C11, BD Biosciences), anti-CD44-PE-Cy7 (clone IM7, BioLegend), and anti-CD62L-BV421 (clone MEL-14, BioLegend). To exclude dead cells from the analysis, a fixable viability stain 510 (BD Biosciences) was used. Samples were analyzed using BD FACS Aria II and the collected data was analyzed using FlowJo software (Tree Star).

The following Abs were used for cell surface staining: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Texas Red (clone 53.6.7), anti-CD44 PE-Cy7 (clone IM7), anti-B220 PE Texas Red (clone RA3-6B2), anti-NK1.1 FITC (clone PK136), anti-CD69 Alexa Fluor 647 (clone H1-2F3), anti-Ly6C APC-Cy7 (AL-21), anti-Ly6G PE-Cy7 (clone 1A8) (Biolegend, San Diego, CA, USA), anti-CD4 APC e-Fluor 780 (clone GK1.5), anti-CD62L PE-Cy5 (clone MEL-14) (eBiosciences, San Diego, CA, USA), anti-CD45RB PE (clone 16A), anti-CD3 Alexa Fluor 647 (clone 17A2), anti-CD4 PerCP (clone RM4-5), anti-CD8 PE (clone 53-6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-CD45 PE (clone 30-F11), anti-CD11c FITC (clone HL3) and anti-CD11b Alexa Fluor 700 (clone M1/70) (BD Biosciences, San José, CA, USA). The abs for intracellular staining included anti-FoxP3 Alexa Fluor 488 (clone MF23), anti-IFNγ Alexa Fluor 700 (clone XMG1.2), TNFα PE-Cy7 (clone MP6-XT22), IL-2 FITC (clone JES6-5H4) (BD Biosciences) and anti-CTLA-4 PE (clone UC10-4F10-11) (Biolegend). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Molecular Probes, Eugene, OR) was used for dead cell exclusion.