Trustain fcx anti mouse cd16 32 antibody clone 93

Blood samples and spleen cell suspensions were incubated with TruStain fcX (anti-mouse CD16/32) antibody (clone 93; isotype Rat IgG2a, λ; diluted 2/100, cat. no. 101319; BioLegend, San Diego, CA, USA) in order to block non-specific antibody binding, then stained for 20 min at room temperature with the following antibodies: Alexa Fluor 647 anti-mouse CD3ε (clone 145-2C11, isotype Armenian Hamster IgG, diluted 0.5/100, cat. no. 100322), PE/Cy7 anti-mouse CD4 (clone GK1.5, isotype Rat IgG2b, κ, diluted 1.25/100, cat. no. 100422), Alexa Fluor 488 anti-mouse CD8a (clone 53–6.7, isotype Rat IgG2a, κ, diluted 0.5/100, cat. no. 100723), PerCP/Cy5.5 anti-mouse CD19 (clone 6D5, isotype Rat IgG2a, κ, diluted 1.25/100, cat. no. 115534) and PE anti-mouse NK1.1 (clone PK136, isotype mouse IgG2a, κ, diluted 1.25/100, cat. no. 108708) (all from BioLegend). Surface staining for blood samples was followed by red blood cell lysis with RBC Lysis Buffer (BioLegend) for 10 min at room temperature in the dark and centrifugation at 350 × g for 5 min, 20°C. Cells were washed twice with Cell Staining Buffer (BioLegend) and analysed by flow cytometry.

Splenocyte single-cell suspensions were prepared by crushing half of a spleen through a 35-μm filter-capped polystyrene tube (Corning) with a 1-ml syringe plunger. Red blood cells were lysed using ammonium potassium chloride lysis buffer (Thermo Fisher Scientific) for 1 min at room temperature, and remaining cells were washed with PBS. The splenocytes were evaluated for viability by staining with LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific), washed, and incubated for 30 min at 4°C with TruStain FcX (anti-mouse CD16/32) antibody (clone 93; BioLegend, San Diego, CA, USA), AF700-conjugated anti-CD19 (clone 1D3; Invitrogen), allophycocyanin (APC)–conjugated anti-CD3 (clone 145-2C11; BioLegend), phycoerythrin/cyanine-7 (PE/Cy7)–conjugated anti-CD86 (clone GL1; Thermo Fisher Scientific), or PE/CF594-conjugated anti-CD69 (clone H1.2F3; BD Biosciences) antibodies. After acquisition, cells were gated on live CD19⁺/CD3⁻ cells, and percentages of CD86⁺/CD69⁺ cells were reported as activated B cells.