We collected the bone marrow cells from the tibiae of rGDF11- or vehicle-treated animals by flushing. Cells were incubated in the red blood cell lysis buffer for 5 min at room temperature. The sorting of BMSCs were performed as described61 (link). Briefly, the cell aliquots were incubated with phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, peridinin chlorophyll protein (Per CP)-, and allophycocyanin (APC)-conjugated antibodies against mouse CD29, Sca-1, CD11b and CD45 (BioLegend, San Diego, CA) at 4 °C for 30 min. Cell sorting was performed on a fluorescence-activated cell sorting (FACS) Aria model (BD Biosciences), and analysis was performed with fluorescence-activated cell sorting DIVE software version 6.1.3 (BD Biosciences).
The sorted Sca-1+CD29+CD45−CD11b− cells were collected and plated into 6-well plates (1,000 cells per well). After 4 h of adhesion, unattached cells were removed. Medium was changed every 3 days, and cultures were fixed and stained with 0.5 mg ml−1 of crystal violet on day 10. Colonies containing 50 or more cells were counted.
The sorted Sca-1+CD29+CD45−CD11b− cells were collected and plated into 6-well plates (1,000 cells per well). After 4 h of adhesion, unattached cells were removed. Medium was changed every 3 days, and cultures were fixed and stained with 0.5 mg ml−1 of crystal violet on day 10. Colonies containing 50 or more cells were counted.