Multiparameter Flow Cytometry Analysis of Diverse Cell Sources

Cells isolated from bone marrow, peripheral blood, and cell lines were stained with multiparameter panels according to the manufacturer's instructions. Briefly, up to 5 × 10⁶ cells were washed in pure phosphate-buffered saline (PBS, Thermo Fisher Scientific), centrifuged at 300 × g, supernatant was decanted, and cells were stained with the indicated antibodies at 4°C for 30 minutes protected from light. After staining, cells were washed with PBS containing 2% FBS (Thermo Fisher Scientific) and resuspended in 100 μL PBS containing 2% FBS for subsequent analysis or sorting. Index sorting for single-cell sequencing was done using a FACSAria Fusion high-speed cell sorter (BD Biosciences) equipped with a 70-μm nozzle as previously described (19, 20 (link)). Antibodies used for index sorting are listed in Supplementary Table S2. Flow cytometry for cell analysis was performed using LSRFortessa (BD Biosciences), Navios (Beckman Coulter), or Aurora (Cytek) instruments.