Igm fitc

Antibodies used for flow cytometry analysis included: CD2-Tri-Color (TC); CD4-Phycoerythrin (PE) and CD4Allophycocyanin (APC); CD28-Fluorescein Isothiocyanate (FITC); CD8-TC; CD19-APC; CD45RA-APC; CD16-FITC from Life Technologies (Grand Island, NY); CD14-PE; CD27-PE; and IgM-FITC from BD Biosciences (San Jose, CA). Freshly thawed PBMCs from each visit were stained with three to five antibodies: T cells (CD2, CD4, CD8, CD45RA, and CD28); B cells (CD19, IgM, and CD27); NK cells (CD16). The data were collected on a BD FACSCalibur or BD FACSCanto II, and analyzed by Cell-Quest (BD Biosciences) and FlowJo. The gating strategies were presented in Additional file 1: Figure S1.

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)).

Peyer’s patches (PP) were isolated from the small intestine and cells were extracted as previously described^{16 (link)}. Briefly, PP were finely cut and incubated for 40 min at room temperature with collagenase/DNase. Cells were then filtered through a 70 µm cell strainer and pelleted by centrifuging at 300×g, 4 °C during 5 min and then resuspended in FACS buffer (PBS, 2% FCS, 5 mM EDTA) and counted before antibody staining. For flow cytometry, cells were first incubated on ice in FACS buffer containing an anti-CD16/CD32 antibody to block the Fc receptor for 10 min and then incubated 30 min on ice in the dark with antibodies against the following surface markers : CD45-PE-eF610 (eBiosciences, clone 30-F11), CD19-PerCp-Cy5.5 (Biolegend, clone 6D5), CD3-PeCy7 (eBiosciences, clone 145-2C11), B220-AF647 (BD Biosciences, clone RA3-6B2), IgD-BV421 (BD Biosciences, clone 11-26c.2a), IgM-FITC (BD Bioscience, clone II/41) and IgA-PE (eBiosciences, clone mA-6E1). Cell viability was evaluated using Fixable Viability Dye eFluor 506 (eBiosciences). Cell acquisition was performed using a CytoFlex (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter).

hESC-derived neural cultures were washed with PBS, trypsinized with 0.05% trypsin for 1–2 min at room temperature and resuspended into single cells using DMEM/F12 with Glutamax containing 10% FBS. For cell surface staining, cells were incubated with CD184-APC (560936; BD Pharmingen), CD24-PE-Cy7 (561646; BD Pharmingen), CD44-PE (51-9007231; BD Pharmingen) and/or CD271-Alexa fluor 647 or BV421 (560877 or 562562; BD Pharmingen) antibodies in PBS containing 1% bovine serum albumen (BSA) according to manufacturer’s protocol. Stained and unstained cells were analyzed using either a BD LSR-II or LSRFortessa flow cytometry machine (BD Biosciences). Control hES9 hESCs or hESC-derived NPCs were used for mCherry control and IgG-PE-Cy7, IgM-FITC, IgG-BV421 and IgG-APC (BD Pharmingen) were used for isotype controls. Cell sorting was performed using either BD FACSAria II or BD Influx cell sorter. Gating for all sorts was defined by isotype control staining. Flow cytometry data analysis was performed using FlowJo software (Tree Star, Inc.).

B cell differentiation in peripheral blood was detected in 100 μl of blood samples washed twice with staining buffer (PBS containing 1% BSA) and stained with a cocktail of mAbs: CD45 V500, CD19 PE-Cy7, CD21 APC, IgM FITC, IgD PE, CD27 PerCP-Cy5.5, and CD38 V450 (all from BD Bioscience) for 20 minutes in the dark at room temperature. Erythrocytes were lysed with BD FACS Lysing solution (BD Bioscience) and washed twice with buffer. B cells were then classified according to their maturation stage into (i) immature (CD21-CD27-) B cells, (ii) naive (CD21+CD27-IgM+) B cells, (iii) memory B cells (CD27+CD19+), (iv) unswitched memory B cells (CD27+IgM+IgD+), (v) memory-switched B cells (CD27+IgM-IgD-), (vi) transitional B cells (CD38+IgM+), and (vii) plasmablasts (CD38+IgM-) (23 (link)) For gating strategy see Supplementary Figure S3, volumes of antibodies per test are shown in Table S1. At least 500,000 cells per sample were collected using a BD Canto II flow cytometer. Data were analyzed using FlowJo 10.8 and BD FACSDiva v8.0.1 software (TreeStar Inc.) (BD Biosciences, San Jose, CA, USA).