Anti cd19 antibody

CD19 expression in skin samples was assessed by immunofluorescence using a specific anti-CD19 antibody (0.5 µg/ml, BioLegend) overnight at 4°C, coupled with DAPI (1:1,000-fold dilution, Thermo Fischer Scientific). Protein visualization was performed by using a specific Alexa Fluor 566 goat anti-mouse antibody incubated for 1 h at room temperature.

The livers and bone marrow cells were harvested from sham and 4T1 cancer-bearing animals 14 days after transplantation. Red blood cells (RBCs) were lysed with RBC lysis buffer (BD Bioscience). The obtained cells were then stained with the following antibodies (BioLegend, CA, USA) for 30 min at 4 °C: anti-CD45 antibody (Clone: 30-F11), anti-CD19 antibody (Clone: 6D5), anti-TCRβ antibody (Clone: H57-597), anti-CD11b antibody (Clone: M1/70), anti-Ly6G antibody (Clone: 1A8), anti-FCεR1a antibody (Clone: MAR-1), anti-CD117 antibody (Clone: 2B8), anti-CD123 (Clone: 5B11), anti-CD49b antibody (Clone: DX5), anti-CD3 antibody (Clone: 17A2), anti-CD11c antibody (Clone: N418), anti-TER-119 antibody, or anti-CD34 antibody (Clone: HM34). Non-viable cells were stained with Propidium Iodide Solution (PI) and gated out. Data were acquired using FACS Aria (BD Bioscience) and analyzed using FlowJo software (v10.8.1, BD Biosciences). Gating strategy is shown in Supplementary Fig. 14.

UCMSCs and BMDMs were isolated with 0.25% trypsin and resuspended in PBS containing 3% FBS for flow cytometry analysis of cell surface markers. UCMSCs were then incubated in the dark for 30 min with phycoerythrin (PE)-conjugated human anti-CD34 antibody (Biolegend, 343506, USA), anti-CD45 antibody (Biolegend, 304058, USA), anti-CD105 antibody (Biolegend, 800504, USA), anti-CD90 antibody (eBioscience, 12-0909-41, USA); allophycocyanin (APC)-conjugated human anti-CD14 antibody (eBioscience, 17-0141-81, USA), anti-CD19 antibody (Biolegend, 302212, USA); and fluorescein isothiocyanate (FITC)-conjugated human anti-CD73 antibody (Biolegend, 344015, USA) and anti-HLA-DR antibody (Biolegend, 307603, USA). BMDMs were incubated with APC-conjugated mouse anti-F4/80 antibody (Biolegend, 123116, USA) and anti-CD11b antibody (Biolegend, 101206, USA) in the dark for 30 min. Associated conjugated immunoglobulins, provided by eBioscience and Biolegend, USA, were used as negative controls. Finally, cells were washed twice in PBS, and flow cytometry was used to detect positive cells (Beckman Coulter, USA).