Cd3 eflour450

SVF pellets were washed with PBS/2% FBS, incubated with Fc Block (BD Bioscience, San Jose, CA, USA) for 15 min and then stained with the following conjugated antibodies (30 min at 4 °C in the dark): CD45-APC-Cy7, CD3-APC, CD19-APC, NK1.1-APC, F4/80-PE-Cy7, CD11b-PerCP-Cy5.5 (all from BD Bioscience), and TER119-APC and CD11c-PE (eBioscience, San Diego, CA, USA) for ATM, CD45-APC-Cy7, CD19-PE, CD4-V500, CD8a-PerCP-Cy5.5, Gr1-APC (BD Bioscience), and CD3-eFlour450, NK1.1-PE-Cy7, TER119-APC, F4/80-APC (eBioscience) for lymphocyte population. After incubation, cells were washed and resuspended in PBS/2% FBS and then analyzed by FACS Canto II (BD Biosciences) and FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA). As described previously [25 (link)], ATM were defined as CD45⁺, CD3⁻, CD19⁻, NK1.1⁻, TER119⁻, CD11b⁺ and F4/80⁺. T cells (CD19⁻CD3⁺NK1.1⁻), B cells (CD19⁺CD3⁻), and NK cells (CD19⁻CD3⁻NK1.1⁺) were gated after excluding the myeloid lineage (Gr1⁺, F4/80⁺) and red blood cells (TER119⁺) among immune cells (CD45⁺).

BM cells were incubated with Fc Block (BD Bioscience) for 15 min, and then stained with the following conjugated antibodies (30 min at 4°C in the dark): CD45-APC-Cy7, NK1.1-APC, CD3-APC, CD19-APC, CD11b-PerCP-Cy5.5, Ly6C-FITC (all from BD Bioscience), TER119-APC, Ly6G-eFluor450 (eBioscience) for neutrophil and monocyte populations, CD45-APC-Cy7, CD19-PE, CD4-V500, CD8-PerCP-Cy5.5, Gr1-APC (BD Bioscience), and CD3-eFlour450, NK1.1-PE-Cy7, TER119-APC (eBioscience) for lymphocyte population. After antibody staining, the cells were incubated with 7-AAD for 5 min at room temperature as a viability dye for dead cell exclusion and analyzed in the presence of AccuCheck counting beads by FACSSymphony A3. The data were further analyzed by using the FlowJo software.