Cd140a pe

For staining of cells from bone marrow and blood, cells were first harvested and incubated with red blood cell lysis buffer (eBioscience, 00-4333) to lyse red blood cells, and passed through a 40-μm cell strainer to obtain single-cell suspensions. Cells were then stained with DAPI (Molecular Probe, D1306) to exclude dead cells from the analysis where indicated, and further analyzed for tdTomato expression. For staining of vascular c-Kit⁺ SPCs, cells were first stained with indicated conjugated-antibodies (1:50) for 30 min on ice. Conjugated-antibodies used in this study include c-Kit-PE (Biolegend, 105807), Sca-1-PE (BD biosciences, 553108), CD31-PE/Cy7 (Biolegend, 102523), CD34-AF647 (Biolegend, 103124), CD29-FITC (Abcam, ab21845), CD45-PerCP/Cy5.5 (Biolegend, 103131), CD105-PE (Biolegend, 120407), CD140a-PE (eBioscience, 12-1401-81), and α-SMA-AF488 (Abcam, ab184675). Corresponding control isotype antibodies were used as control. For analyses of glucose uptake, 2-NBDG (100 μM, Invitrogen, N13195) was added to cultured cells and incubated for 1 h at 37 °C. Cells were then washed with PBS, detached by trypsin, and suspended in PBS for further analysis. All prepared samples were analyzed by a BD LSR Fortessa II flow cytometer (Becton Dickinson) or BD ACCURI C6 flow cytometer (BD Biosciences). FlowJo v10 software (Tree Star) was used to analyze the flow cytometric data.