Anti gl 7 biotin

Single cell suspensions were prepared from spleens. Red blood cells were lysed in hypotonic NH₄Cl and stainings were performed at 4°C in PBS+4% FCS and 1 mM EDTA. Briefly, cells were blocked by 10 min incubation with FcBlock (anti-CD16/32, 2.4G2, BD bioscience), washed, and stained for 20 min. For biotinilated antibodies, extra 20 min incubation with streptavidin was performed. MuHV-4 infected cells were monitored based on their endogenous YFP expression. The following antibodies were used: anti-CD69 PE (H1.2F3), anti-CD95 PE (Jo2), anti-CD19 APC-Cy7 or APC-H7 (1D3), anti-IgM PE (R6-60.2), anti-IgD Biotin (11-26c.2a), CD11b PE or v450 (M1/70) (BD Biosciences); CD45.2 Brilliant Violet 510 (104) (BioLegend); anti-CD45.1 PeCy7 (A20), and anti-GL-7 Biotin (Ebioscience). Streptavidin-Cy5 (BD Biosciences) was used to reveal biotinilated antibodies. HEL specific B cells were identified with the HEL-A647 conjugate described above. Samples were acquired on a FACS Canto or on a LSR Fortessa (BD Biosciences), using DIVA software (BD Biosciences) for acquisition and Flowjo v.6.4.7 (Tree Star) for analysis. Cells were gated on live cells based on FSC/SSC parameters and cell doublets were excluded based on FSC-W signal.