Gr1 af700

Myeloid cells were extracted from whole hemispheres, isolated into single-cell suspensions and identified using fluorescence-activated cell sorting (FACS) gating for CD11b⁺CD45^int as previously described (Elmore et al., 2014 (link)). Cells were stained with the following surface antibodies purchased from Biolegend (San Deigo, CA) at 1:200 unless otherwise indicated: CD34-eFlour660 (1:50, 50-0341-80, eBioscience, San Diego, CA), Sca-1-AF700 (1:100, 108141), CD16/32-PE (101307), Ter119-PE/Cy5 (116209), ckit/CD117-PE/Cy7 (25-1171-81, eBioscience), CD150/SLAM-PerCP-eFlour710 (46-1502-82, eBioscience), CD11b-APC (101212), CD11b-PE (101208), Gr1-AF700 (108422), CD45-AF700 (103128), CD45-APC/Cy7 (103116), NK1.1-PE (108707), CD3-PE/Cy7 (100220), CD19-Per-Cyanine5.5 (45-0193-82, eBioscience), CD11c-APC/Cy7 (117323), Ly6C-PE (1:400, 128007), Ly6G-5.5 (127615). For HSCs, CMPs, and GMPs, all cells were gated on live (PI^-), Ter119^- cells and then identified with the following gating strategy: HSCs: FcyR^-, ckit⁺ Sca⁺ CD34^-, SLAM⁺, CMPs: FcyR^-, ckit⁺, Sca^-, CD35⁺, and GMPs: FcyR⁺, ckit⁺, Sca^-, CD34⁺. Samples were acquired with the BD LSRII or BD Fortessa X20, and sorted with the BD FACS Aria II.

Bone marrow/hematopoietic stem cells were extracted from femurs and tibia by flushing with PBS + 2% FBS. Peripheral blood cells/granulocytes were collected via the tail vein in EDTA. Red blood cells were lysed using 1x ACK Lysis Buffer. Cells were then stained for analysis by flow cytometry with the following surface antibodies purchased from Biolegend at 1:200 unless otherwise indicated: CD34-eFlour660 (1:50, 50-0341-80, eBioscience), Sca-1-AF700 (1:100, 108141), Ter119-PE/Cy5 (116209), ckit/CD117-PE/Cy7 (25-1171-81, eBioscience), CD150/SLAM-PerCP-eFlour710 (46-1502-82, eBioscience), CD11b-APC (101212), Gr1-AF700 (108422), CD45-APC/Cy7 (103116), NK1.1-PE (108707), and CD27-APC/Cy7 (124226). Flow cytometry analysis was performed using the BD LSRII.