Anti cd62l mel 14

Stained cells were analyzed using an LSRII system (BD Biosciences). Data were analyzed with FlowJo V10 (TreeStar). Cell viability was evaluated using SYTOX Blue or DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Life Technologies). The following antibodies were used: anti-CD90.1 (HIS51), anti-CD4 (RM4-5), anti-CD8α (53–6.7), anti-CD6 (REA311), anti-TCRβ (H57-597), anti-γδ TCR (GL-3), anti-CD3e (145-2C11), anti-CD44 (IM7), anti-CD25 (PC61), anti-CD45R (RA3-6B2), anti-CD5 (53–7.3), anti-CD62L (MEL-14), and anti-human heparin-binding epidermal growth factor (HB-EGF; BAF259) from BD Biosciences, Miltenyi, R&D Systems, and eBioscience. Note that the human heparin-binding epidermal growth factor constitutes the receptor for diphtheria toxin.

Spleen cells were isolated as described above and placed in ice-cold PBS supplemented with 5% FCS and 0.1% sodium azide. Staining was performed as previously described [11 (link)]. The following fluorochrome-conjugated monoclonal antibodies were used: anti-CD4 [H129.19]; anti-CD8 [53-6.7]; anti-CD19 [MB19-1]; anti-CD25 [7D4]; anti-CD44 [IM7]; anti-CD69 [H1.2F3]; anti-Gr-1/Ly6C/Ly6G [RB6-8C5]; anti-CD45RB [16A]; anti-CD62L [MEL-14]; anti-CD11b [M1/70] (BD Pharmingen, San Diego, CA); and anti-CD11c [HL3]—(Serotech, Raleigh, NC) anti-F4/80 [CI:A3-1] and anti-GITR [DTA-1] (eBioscience, San Diego, CA). After staining, the cells were fixed with 1% paraformaldehyde in PBS and analyzed using a FACSCanto (BD Biosciences, San Jose, CA), 50,000 events/sample recorded. Data were processed using FlowJo software (FlowJo LLC, Ashland, OR). Cell numbers were calculated using the percentage obtained by FACS analysis and the total numbers of leukocytes counted in a hemocytometer.

Spleens were aseptically isolated from ten mice from each group 4 weeks after challenge. Splenocyte suspensions were prepared in RPMI-1640 culture media supplemented with 10% FBS, after RBC lysis with red blood cell lysing buffer hybrid-max (Sigma-Aldrich, St. Louis, MO, USA), for flow cytometry analysis, antibody secreting cell (ASC) assays and cytokine analysis. Cells were stained with trypan blue (Welgene, Daegu, South Korea) and counted with a hemocytometer chamber under a microscope. Splenocytes from each animal were resuspended in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS). Cells from individual mouse were separately incubated with Fc Block (clone 2.4G2; BD Biosciences, CA, USA) to block non-specific binding at 4°C for 15 min, and then stained at 4°C for 30 min with different combinations of FITC, PE, PE-Cy5, PE-Cy7 or APC conjugated anti-CD3e (145-2c11), anti-CD4 (GK1.5), anti-CD8a (53–6.7) (BD Biosciences, CA, USA). For memory T cell responses, anti-CD44 (IM7) and anti-CD62L (MEL-14) were used (BD Biosciences, CA, USA) as indicated [28 ]. For memory B cell responses, anti-CD45R/B220 (RA3-6B2), anti-CD27 (LG-3A10) and anti-IgG1 (A85-1) were used (BD Biosciences, CA, USA) [29 (link)]. Events were acquired on BD Accuri C6 Flow Cytometer (BD Biosciences, CA, USA) and data were analyzed using C6 Analysis software (BD Biosciences, CA, USA).