Freshly isolated bone marrow cells were resuspended in ice-cold FACS buffer (PBS added with 2% FBS) containing a mix of surface marker-specific antibodies carrying different fluorescent tags. For BMSC detection, the following BD Biosciences antibodies were used: anti-cd45 PerCP, anti-cd31 PerCP-eFluor 710, anti-cd105 eFluor450, and anti-cd29 APC, all diluted 1:500. For hematopoietic cell analysis, the following BD Biosciences antibodies were used: anti-cd45 APC Cy7, anti-cdllb AF700, anti-Ly6C BV605, anti-Ly6G BV786, and anti-F4/80 PE-Cy5, all diluted 1:100.
Cells were incubated at +4°C with agitation for 30 min with the above antibodies, centrifuged in a table-top centrifuge at 7000 rpm for 1 minute, washed with 500 μL ice-cold FACS buffer, centrifuged again, and finally resuspended in 250 μL ice-cold FACS buffer for analysis. Flow cytometry was performed at the University of Rochester Core Facility using BD Biosciences LSRII flow cytometer.
Cells were incubated at +4°C with agitation for 30 min with the above antibodies, centrifuged in a table-top centrifuge at 7000 rpm for 1 minute, washed with 500 μL ice-cold FACS buffer, centrifuged again, and finally resuspended in 250 μL ice-cold FACS buffer for analysis. Flow cytometry was performed at the University of Rochester Core Facility using BD Biosciences LSRII flow cytometer.