Pe anti mouse cd34

Bone marrow cells were harvested from mouse femurs and tibias and washed with PBS. The cells were then treated with 5% rat serum for 15 minutes at 4°C, followed by incubation with the primary antibody for 30 minutes at 4°C in the dark. After rinsing with PBS the next day, the cells were stained with Flexible Viability Dye and fixed with 1% paraformaldehyde (PFA). The following antibody cocktail was used: BV421 anti-mouse lineage cocktail, Biolegend, cat#: 133311; PE-CF594 anti-mouse CD127, BD Biosciences, cat#: 562419; PE/Cy7 anti-mouse Sca-1, Biolegend, cat#: 108114; FITC anti-mouse c-Kit, Biolegend, San Diego, CA, cat#: 105806; PE anti-mouse CD34, Biolegend, cat#: 119; PerCP-eFluor 710 anti-mouse CD135, eBioscience, San Diego, CA, cat#: 46–1351-82; and APC anti-mouse CD16/CD32, eBioscience, cat#: 14–0161-82.

The cell surface markers of the BMMSCs and Mφs were analyzed by flow cytometry. Briefly, the cells were trypsinized and washed with PBS. To block Fc receptors, the cells were incubated with 2% anti-mouse CD16/32 (BioLegend, San Diego, CA, USA) on ice for 10 min. Then, the cells were washed twice with PBS and incubated with specific antibodies for 30 min at 4 °C in the dark. Excess antibody was removed by washing the cells with PBS. Untreated cells were used as blank controls. The samples were then analyzed with a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, USA). For the characterization of BMMSCs, the following antibodies were used: PE anti-mouse Ly-6A/E (Sca-1), PE anti-mouse CD90.2, PE-anti-mouse CD73, PE anti-mouse CD105, PE anti-mouse CD34 and FITC anti-mouse CD45 (all from BioLegend, San Diego, CA, USA). FITC anti-mouse CD86 and PE anti-mouse CD206 (both from BioLegend, San Diego, CA, USA) were used for the identification of Mφs. In this experiment, pulse width measurements were used to eliminate the possibility that the detected cells were doublets of RAW 267 cells. 7-aminoactinomycin D (7-AAD), as well as isotype controls were used to exclude dead cells and the nonspecific binding of the monoclonal antibodies. Each group with no less than 10⁶ cells was gated for flow cytometric analysis.