Anti human cd19 hib19

Non-specific antibody binding was blocked by incubating cells with TruStain FcX (eBioscience, San Diego, CA, USA) on ice for 15 minutes. The cells were subsequently incubated with monoclonal antibodies (Table 1) on ice for 30 minutes. The monoclonal antibodies included anti-human CD45 (HI30, BioLegend, San Diego, CA, USA) for whole leukocytes, anti-human CD3 (HIT3a, BD Pharmingen, San Jose, CA, USA) for T cells, anti-human CD14 (HCD14, M5E2, BioLegend, San Diego, CA, USA) for macrophages, anti-human CD19 (HIB19, BioLegend, San Diego, CA, USA) for B cells, anti-human CD11c (3.9, BioLegend, San Diego, CA, USA) for dendritic cells, anti-human CD16 (3G8, BioLegend, San Diego, CA, USA) for granulocytes including neutrophils and eosinophils, and anti-human HLA-DR (L243, BioLegend, LN3, eBioscience, San Diego, CA, USA) for dendritic cells and B cells. Flow cytometry was performed using a FACS Canto II (BD Biosciences, San Jose, CA, USA) and data were analyzed using Flow Jo ver. 9.3.2 (Treestar, Inc., San Carlos, CA, USA) (Fig 1).

For FACScan analysis and screening of different gene knockout (KO) cells, the following anti‐human antibodies were used: anti‐CD79A‐PE (HM47, BioLegend), anti‐CD79B‐PE (CB3‐1, BioLegend), anti‐IgM‐APC (G20‐127, BD Biosciences), mouse IgG1, κ isotype control‐APC (MOPC‐21, BD Biosciences), anti‐IgD‐PE (IA6‐2, BD Biosciences), mouse IgG2a, κ isotype control‐PE (G155‐178, BD Biosciences), anti‐Igλ‐APC (1‐155‐2, eBioscience), anti‐CD19‐Alexa fluor 647 (HIB19, BioLegend), and anti‐CD81‐APC (1D6, eBioscience). To stimulate cells and to measure calcium responses, anti‐CD79B (CB3‐1, BioLegend), anti‐CD79B (EPR6860, Abcam), and anti‐CD19‐Alexa fluor 647 (HIB19, BioLegend) antibodies were used. To prepare PLA probes and F(ab′)₂ fragments, the following antibodies were used: anti‐human CD79B (CB3‐1, Acris), anti‐human CD19 (HIB19, Biolegend). The following antibodies were used for Western blot (WB): anti‐IgM (Southern Biotech), anti‐IgD (Southern Biotech), anti‐lambda (Southern Biotech), anti‐CD79A (HM47, BioLegend), anti‐CD79B (CB3‐1, Southern Biotech), anti‐BLNK (2B11, Santa Cruz Biotechnology), and anti‐GAPDH (6C5, Thermo Fisher).