Igm bv510

Cells grown in EHT culture or harvested animal tissues were stained with the following antibody panels. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter), CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD), TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD), and CD31/PECAM PE (WM59; BD). All stains were performed with fewer than 1 × 10⁶ cells per 100 μl staining buffer (PBS + 2% FBS) with 1:100 dilution of each antibody, for 30 min at 4 °C in the dark. Compensation was performed by automated compensation with anti-mouse Igk negative beads (BD) and cord blood MNC stained with individual antibodies. All acquisitions were performed on a BD Fortessa cytometer. For detection of engraftment, human cord-blood-engrafted mouse marrow was used as a control to set gating; sorting was performed on a BD FACS Aria II cell sorter.

Peripheral blood mononuclear cells (PBMCs) were acquired and then were stained for surface markers for 20 min in PBS containing fluorescent antibodies. Fluorescently labeled antibodies included CD3-PerCP-Cy5.5, CD25-PE, CD45RA-FITC, CD8-PerCP-Cy5.5, CD19-PerCP-Cy5.5 and CD56-PE-Cy7 (Tianjin Three arrows, China); CD4-APC-H7, CD8-BV510, CD127-BV421, CCR7-AF647, CD28-PE-Cy7, CD3-APC-H7, CD4-PE-Cy7, CXCR3-Alexa488, CXCR6-BV510, CXCR5-Alexa647, CCR4- BV421, PD-1-PE, CD45-APC-H7, CD27-BV421, IgD-BB515, IgM-BV510, CD38-APC, CD24-PE, and CD21-PE-Cy7 (BD, United States). The instrument settings and gating strategies were adopted from previous works (Supplementary Figure S1) (Yang et al., 2020 (link); Zhu et al., 2020 (link)). All experiments, including cell separation and sample preparation, were performed according to standardized experimental manuals. Samples were analyzed using CytoFLEX flow cytometer (Beckman, United States). Results are expressed as the proportion of cells expressing particular markers. T cell subsets, including cytotoxic T (Tc) cells, helper T (Th) cells, T follicular helper (Tfh) cells, regulatory T (Treg) cells, B cells and NK subsets were identified (Supplementary Table S1).