Cd20 af700

B cells were isolated from PBMCs using CD19 microbeads (Miltenyi Biotec) and stained with DAPI (Thermo Fisher), CD20-AF 700, IgG-APC, IgD-Pe-Cy7, IgM-FITC, and CD27-PE (all BD Biosciences) for 30 min on ice. 200,000 CD20⁺IgG⁺IgM^-IgD^-CD27^- B cells were sorted into FBS (Sigma-Aldrich) using a BD FACSAria Fusion, and RNA of sorted B cells was isolated with the RNeasy Micro Kit (QIAGEN). cDNA was generated by template-switch reverse transcription according to the SMARTer RACE 5′/3′ manual using the SMARTScribe Reverse Transcriptase (Takara) with a template-switch oligo including an 18-nucleotide unique molecular identifier. Heavy-chain variable regions were amplified with an IgG-specific nested PCR and amplicons were used for library preparation and MiSeq 2x300 bp sequencing (Illumina). Raw NGS reads were pre-processed and assembled to final sequences as previously described (Ehrhardt et al., 2019 (link)).

Whole blood was lysed by diluting in lysis buffer (1x) (Biolegend) and washed in cold FACS buffer to retrieve a leukocyte pellet. Cell populations were distinguished by fluorescently labelling using a previously optimised polychromatic staining panel; LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Invitrogen), CD18 PECy5, CD66b PERCPCy5.5, CD14 BV421, HLA DR BV510, CD15 605, CD20 AF700, CD19 APC Cy7, HLA DM PE, CD3 PECF594, and HLA ABC (BD Biosciences). Relevant fluorescence minus one (FMO) controls and negative controls were used to identify positively stained populations. Lymphocytes, monocytes and granulocytes were identified by size forward scattered light (FSC)/ side scattered light (SC]) and then further phenotyped into live CD3− CD14+ monocytes, CD3+ lymphocytes, CD3− CD19/CD20 expressing B cells and CD66b/CD15 positive neutrophils. Flow cytometry was performed on the Fortessa instrument (BD Biosciences) and data analysed using ‘Flow Jo’ (Tree Star) software.

Mock- or MCM-treated immortalized B cells or TAB were stained with the following antibodies or matched isotypes and analyzed on a FACS Aria III (BD): CD19 BV711 (clone SJ25C1, 0.06 µg/100 µl, catalog number 563036), CD20 AF700 (clone 2H7, 0.5 µg/100 µl, 560631), CD24 PE-CF594 (clone ML5, 1 µg/100 µl, 562405), CD27 BV421 (clone M-T271, 0.25 µg/100 µl, 562513), CD38 APC (clone HIT2, 0.125 µg/100 µl, 555462), CD138 PE (clone MI15, 0.125 µg/100 µl, 552026), IgD PE-Cy7 (clone IA6-2, 0.125 µg/100 µl, 561314), IgG FITC (clone G18-145, 0.125 µg/100 µl, 555786), IgM BV605 (clone G20-127, 0.5 µg/100 µl, 562977) (all BD biosciences). Live/dead cell exclusion was performed by addition of 7-AAD (5 µg/ml, Calbiochem) prior to acquisition of the samples. Data were analyzed using FlowJo 10.4.2 (FlowJo LLC). The gating strategy is shown in Supplementary Fig. 10.