Cd11b

Uteri were removed from mice, cut longitudinally, and then cut into ~0.25 cm sections and placed in 10× Gentle Collagenase/Hyaluronidase (Stem Cell Technologies #07919) diluted 1:10 in DMEM F12 (Gibco #12634-010) for 4 h at 37°C with intermittent vortexing. Digested uteri were centrifuged for 5 min at 450g, resuspended in ammonium chloride (Stem Cell Technologies #07800) to lyse the red blood cells, and washed with PBS. Digested uteri were centrifuged again, resuspend in Trypsin EDTA (Stem Cell Technologies #07901), triturated prior to adding HBSS + 2% FBS (HF), recentrifuged and resuspended in Dispase with 1 mg/ml DNaseI (Stem Cell Technologies #07913 & #07900) by trituration. Cells were resuspended in HF and 0.1 mg/ml DNaseI and incubated with EpCAM (Biolegend #118217), CD11b (TONBO Biosciences #20-0112) and Thy1 (Biolegend #105325) antibodies for 30 min at 4°C in the dark. After staining cells were washed two times in HF and resuspended in HF with DNaseI with DAPI. Data was acquired using Beckman Coulter MoFlow Astrios and at least 500 000 events were collected in the live, single-cell gates for experimental tubes and 200 000 events for control tubes. Fluorescence minus one controls were used to set gates and single color tubes for both antibodies and endogenous fluorescence were used for compensation. Data were analyzed using Beckman Coulter Kaluza Analysis software.

Bone marrow cells were flushed from the femurs and tibias of 2-month-old mice (C57BL/6J) and incubated for 2 min at room temperature with BD Pharm Lyse hypotonic lysis buffer (BioLegend, 420301) for red blood cell (RBC) lysis. Cells were washed twice with cold fluorescence-activated cell sorting (FACS) buffer, incubated with Fc blocking buffer (BD Biosciences, 564765) for 15 min at 4°C, and treated with antibody cocktail including CD11b (Tonbo Biosciences, 20-0112), CD45R/B220 (Tonbo Biosciences, 65-0452), CD117 (Tonbo Biosciences, 60-1172), CD3 (Tonbo Biosciences, 50-0031), Ter119 (BioLegend, 116233), and Ly6C (BioLegend, 128017) in cold FACS buffer. After treatment with DAPI, cells were subjected to FACS analysis using a BD LSR II flow cytometer (BD Biosciences). The data were analyzed using FlowJo (v.10.1).