Anti igd pe ia6 2

For cell death analysis PBMCs were washed in FACS buffer. Fc-receptors were blocked with anti-IgG (Jackson IR). Cells were incubated for 15 min at RT in binding buffer containing anti-CD4-APC (RPA-T4, eBioscience) and anti-CD8-PacificBlue (DK25, Dako) antibodies for cell surface staining as well as FITC Annexin V (BD Pharmingen). To discriminate between viable cells, cells that were in early apoptosis and cells that were in late apoptosis or already dead 1 mg/ml of the vital dye PI (Sigma Aldrich) was added 5 min prior to cell acquisition by flow cytometry. To investigate the influence of edelfosine on MHC class II expression by human B and T cells, cells were stained for viability by using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). Cells were washed with PBS, blocked and stained for cell surface antigen expression as described before. Antibodies were anti-CD3-PE-Cy7 (UCHT1), anti-CD4-APC (RPA-T4), both from eBioscience, anti-CD8-PacificBlue (DK25, Dako), anti-CD5-PerCP-Cy5.5 (L17F12), anti-CD19-V450 (HIB19), anti-IgD-PE (IA6-2) and anti-HLA-DR/DP/DQ-FITC (Tu39), all from BD Biosciences, as well as anti-CD27-APC-Alexa750 (CLB-27/1) and anti-CD45RA-PE-Cy5.5 (MEM-56), both from Invitrogen. Data was acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with FACSDiva (BD Biosciences) and FlowJo (Tree Star) software.

For surface staining of Ramos cells, the following anti-human antibodies, aptamers, and nanobodies were used: anti-IgM eFluor 450 (SA-DA4, eBioscience, 12-9998-42), TD05 Cy5, TD05+ Cy5, TD05− Cy5, TD05 Cy3 (custom order from Sigma-Aldrich), Enh Cy5 (homemade), and anti-IgD PE (IA6-2, BD Bioscience, 555779). For surface staining of mouse splenic B cells, the following anti-mouse antibodies were used: anti-IgM eFluor 450 (eB121-15F9, eBioscience, 14-5890-82) and anti-IgD FITC (11-26c.2a, Biolegend, 405704). For stimulating mouse splenic B cells, anti-IgM (polyclonal, SouthernBiotech, 2020–01) and anti-IgD (polyclonal, eBioscience, 24-5093-51) were used. For bPHA probe preparation, the following antibodies were used: anti-CD79a (JCB117+HM47/A9, abcam, ab213114) and anti-Syk (SYK-01, Biolegend, 626202).

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).