Cd38 bb515

PBMCs were thawed and B cells were enriched using EasySep™ pan B cell magnetic enrichment kit (STEMCELL). B cells were stained with a panel containing CD19 PE-Cy7 (Biolegend), IgM APC (Southern Biotech), CD27 BV605 (Biolegend), CD38 BB515 (BD Biosciences), and CD3 BV510 (BD Biosciences). B cells were stained with surface stain master mix and each COVID-19 antigen probe for 30 minutes on ice in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin. Cells were stained with probe at a 1:100 dilution (NP, ORF7a, ORF8, RBD) or 1:200 dilution (spike). Cells were subsequently washed with 1X PBS 0.2% BSA and stained with Live/Dead BV510 (Thermo Fisher) in 1X PBS for 15 minutes. Cells were washed again and re-suspended at a maximum of 4 million cells/mL in 1X PBS supplemented with 0.2% BSA and 2 mM Pierce Biotin for downstream cell sorting using the MACSQuantTyto cartridge sorting platform (Miltenyi). Cells that were viable/CD19⁺/antigen-PE⁺ were sorted as probe positive. The PE⁺ gate was drawn by use of FMO controls. Cells were then collected from the cartridge sorting chamber and used for downstream 10X Genomics analysis.

Urine and blood samples were collected from ten AAV patients with active disease. Urine samples were prepared as described previously (11 (link)). Briefly, urine was diluted 1:1 in PBS and centrifuged at 1,800 rpm. The sediment was resuspended in PBS and mononuclear cells (MNCs) were isolated using lymphoprep (Axis-Shield, Oslo, Norway). Next, MNCs were resuspended in wash buffer and stained with anti-human CD19-PerCP-Cy5.5, CD45-BV605, CD27-APC (BioLegend, San Diego, CA, USA), CD3-BUV395, and CD38-BB515 (BD Biosciences) for 15 min at room temperature in the dark. Isotype-matched non-specific antibodies were used as negative controls. In parallel, blood samples were labeled with the aforementioned monoclonal antibodies. Afterwards, cells were treated with 10x diluted FACS lysing solution for 10 min, washed twice in wash buffer and immediately analyzed. Stained urine and blood samples were acquired on the LSR-II and data was analyzed using Kaluza 1.5a software. Figure 3A shows a representative gating example of both blood and urine. Three patients were excluded because no renal involvement was diagnosed and accordingly no B cells were present in the urine.