Anti sca 1 pe

A single-cell suspension of isolated Lin⁻BMCs was stained using the Mouse Mesenchymal Stromal Cell 4-Color Flow Cytometry Kit (R&D, Minneapolis, MN, USA), containing conjugated antibodies to CD105-FITC, CD29-PE, Sca-1-APC, and CD45-PerCP. To expand the analysis, some of the samples were also stained with the following antibodies: anti-CD90-APC (R&D, Minneapolis, MN, USA), anti-CD34-APC (BioLegends, San Diego, CA, USA), and anti-Sca-1-PE (BD Biosciences Pharmingen, San Diego, USA). The cells were diluted in 100 μL Staining Buffer (R&D, Minneapolis, MN, USA) and incubated with the antibodies for 45 minutes at 2–8°C. Samples stained with the appropriate isotype control were examined in parallel. After washing, cells were resuspended in fresh Staining Buffer and analyzed by fluorescence-activated analyzer LSRII (BD Biosciences, San Jose, CA, USA). At least 10⁵ events were acquired and analyzed using FACS Diva software (BD Biosciences). The number of cells in each population was expressed as the percentage of the total number of events.

For surface marker analysis, MSCs were harvested and incubated with specific phycoerythrin (PE)-labeled antibodies for 1 hour. Antibodies anti-CD11b–PE, anti-CD45.2–PE, anti-CD31–PE, anti–Ter-119–PE and anti-CD34-PE, rat IgG2b isotype control–PE and rat and mouse IgG2a isotype controls–PE were purchased from eBioscience (eBioscience, San Diego, California, USA), anti–SCA-1–PE was purchased from PharMingen (San Diego, California, USA), and anti–CD34–PE was purchased from Santa Cruz (Santa Cruz, Dallas, Texas, USA). Cells were analyzed using the LSRII flow cytometer (Becton, Dickinson, New Jersey, USA).
For DNA content estimation, cells were fixed with 70% ethanol/phosphate-buffered saline, treated with RNaseA 0.4 mg/ml (Sigma), and stained with propidium iodide 0.1 mg/ml (Sigma). Labeled cells were analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Fresh splenocytes were used as control diploid cells.

The phenotype of MSCs was evaluated at 72h after the second transduction. MSCs were immunolabelled with the following monoclonal antibodies: anti-CD45 (APC-Cy7 or FITC, clone: 30-F11, BD Bioscence), anti-Sca-1 (PE, clone: E13-161.7, BD Bioscence), anti-CD105 (PE/Cy7, clone: MJ7/18, BioLegend), and anti-CD90.2 (APC, clone: 30-H12, BioLegend). Staining was performed according to manufacturer’s protocols for 30 min at 4°C. Cells were analyzed using LSR II flow cytometer and FACS Diva software (Becton Dickinson).

BM-MNC (1.5 × 10⁶ cells/sample) were stained with appropriate conjugated antibodies in PBS with 2% BSA. Specifically, human BM-MNC were stained with anti-CD33-PE-Cy7, anti-CD34-PerCP-Cy5.5, anti-CD14-BV510, anti-CD71-BV421, anti-CD38-BV711, anti-CD235a-BUV395, anti-PD-1-FITC, and anti-PD-L1-APC (BD Biosciences). Gating strategies for the identification of myeloid cells, HSPCs, and erythroid progenitors are shown in Supplementary Figure S1. Mouse BM cells were stained with anti-c-Kit-PerCP-Cy5.5, anti-Sca-1-PE, Lin-APC (including anti-CD3ε, anti-CD11b, anti-CD45R/B220, anti-TER-119, anti-Gr-1), anti-PD-1-BV421, anti-PD-L1-BV711, anti-PD-L2-BUV395, and anti-CD16/32-PE-Cy7 (BD Biosciences). Cell viability was determined using near-infrared live/dead dye (BD Biosciences) and the negative (live cell) population was used for further analysis. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo 9.9.3 software. Intracellular staining with PE-conjugated anti-active caspase-3 was performed using the Cytofix/Cytoperm™ protocol following initial cell surface receptor staining (BD Biosciences). For PD-1/PD-L1 ligation experiments, 2 million BM-MNC were plated per well in 24-well plates coated with recombinant human PD-L1 (2 μg/mL) for 24 h at 4 °C.

Flow cytometry analyses of cultured CPC were performed with a BD LSRII flow cytometer. Briefly, CPC cells were blocked with 5% rat serum and stained with a panel of conjugated antibodies, including anti-Sca-1-PE, anti-CD31-biotin,, anti-c-kit-biotin, anti-CD45-PE-cy7, anti-Flk1-PerCP-cy5.5 and anti-CD105-V450 (BD Biosciences, San Jose, CA), or isotype-matched control antibodies. Anti-c-kit-biotin, and anti-CD31-biotin antibodies were resolved via secondary staining with streptavidin-PerCP-Cy5.5 or streptavidin-FITC, respectively.