Fluorescein isothiocyanate fitc conjugated cd45

MSCs at the fourth passage were trypsinized, washed using phosphate buffersaline (PBS) and resuspended in PBScontainingFBS (1%). A 100 μL aliquot of suspended MSCs was incubated for 45 minutes at 4°Cwith one of the following anti-human monoclonal antibodies (mAb): phycoerythrin (PE)-conjugated CD105, CD34, and CD73, or fluorescein isothiocyanate (FITC)-conjugated CD45 (BioLegend, USA). In addition, the mouse isotypic antibodies, including PE-IgG1j and FITC-IgG1j (BioLegend, USA), were utilized as control. After labeling of cells, they were evaluated using BD FACS Calibur™ flow cytometer (BD, USA) and analyzed using Flow Jo 7.6 Software.

Femurs at both the proximal and distal ends of the bones were made small cuts carefully. Whole BM cells were extracted from the femurs by using a 25G needle to flush with a DEME medium containing 10% FBS [19 (link)]. The washes were collected from each of the 4 groups and filtered through a 70 μm cell strainer cap (Corning Falcon, USA) into a fresh 5 ml polypropylene tube [20 (link)]. To isolate native BMSC subsets from mouse BM cells, up to 10⁶ cells were incubated with 20 μL of fluorescein isothiocyanate (FITC)-conjugated CD45 (Biolegend, CA, USA) and 20 μL phycoerythrin (PE)-conjugated CD44 (Biolegend, CA, USA) for 20 min at room temperature avoiding light, washed in FACS buffer, and centrifuged (5 min at 500×g) [21 (link)]. Sorting gates were created by selecting CD45-CD44⁺ cells using a BD FACS Aria II (BD Biosciences) with a 100 μm nozzle (Figure 1). Sort-purified BMSCs were collected into PBS containing 30% FBS and centrifuged (15 min at 1000×g). Mice sort-purified BMSCs were collected and stored at −80°C for further analysis.