Red blood cell lysing buffer hybrid max

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).

Mouse spleens were collected and placed in 1% (v/v) FCS in PBS (1% FCS/PBS). Spleens were then mechanically processed through a 100 μm EASYstrainer Cell Strainer (Greiner Bio-One) using the back of a 5 ml syringe plunger (Terumo Medical). Cells were resuspended in 1% FCS/PBS and centrifuged at 350g before being lysed with 1 ml Red Blood Cell Lysing Buffer Hybrid-Max (Sigma Aldrich) for 5 minutes at RT. Cells were then washed with 10 ml of 1% FCS/PBS, resuspended in 5 ml 1% FCS/PBS, and stored at 4°C until required.