Kdr pe

Fat samples were washed and minced in a sterile petridish with PBS to prevent dehydration. Following digested in 1mg/ml type II collagenase (Sigma, Saint Louis, USA) at 37°C under gentle agitation, the cells were passed through a 70µm pore-size sterile nylon mesh filter (Falcon, Franklin Lakes, USA). Then, the cells were harvested after centrifugation at 200 g for 8 minutes. To remove remaining tissue debris, the pellet was resuspended and filtered through a 40 µm cell strainer. Cells were counted and seeded in culture flasks. The culture medium was changed twice a week. Cells were trypsinized, centrifuged at 500 g for 5 minutes and re-seeded when confluent.
We performed flow cytometry analysis w to verify the cultured ADSCs. In brief, the cultured cells were washed and incubated in blocking buffer for 30 minutes at 4 °C. After being washed, the cells were then incubated for 30 minutes at 4 °C in dark with the fluorescein isothiocyanate (FITC)-conjugated antibodies or thephycoerythrin (PE)-conjugated antibodies as follows: c-kit/FITC, CD9/FITC, CD31/FITC, CD34/FITC, CD90/FITC, CD271/FITC, MAP-2/FITC,VEGF/FITC, KDR/PE, CD29/PE, CD45/PE (BD Biosciences, NJ, USA). To fix the cells, 1% paraformaldehyde was used. Isotype-identical antibodies (IgG) were used as controls. Cell viability of each group was greater than 96.0%. Sample assessment was performed in three times.

Adherent cells were washed with Dulbecco's PBS (DPBS) (Thermo Fisher, Waltham, MA, #12563011) and treated with Tryp‐LE Select (Thermo Fisher, Waltham, MA, #12563011) at 37°C for 5 minutes, followed by gentle pipetting to complete cell dissociation. The dissociated cells were then washed with DPBS several times before antibody staining for flow cytometry. For suspension hematopoietic cells, gentle pipetting was used to dislodge any loosely adhered hematopoietic cells from the adherent stromal layer, and the cells were washed multiple times with PBS before staining. Cells were stained in ice‐cold fluorescence‐activated cell sorting buffer (PBS, 1% BSA). Antibodies used; KDR‐PE (BD Pharmingen, San Jose, CA, #560872), CD34‐APC (BD, San Jose, CA, #555824), CD144‐PE (BD, San Jose, CA, #561714), CD45‐PE (BD, San Jose, CA, #561866), CD117‐APC (Thermo Fisher, Waltham, MA, clone 104D2), CD117‐PE (BD, San Jose, CA, #555714), CD235a‐FITC/PE (BD, San Jose, CA, Clone GA‐R2 (HIR2)), CD41a‐APC (BD, San Jose, CA, #559777), CD71‐FITC (BD, San Jose, CA, #555536), CD73‐PE (BD, San Jose, CA, #561014), CXCR4‐Biotin (BD, San Jose, CA, #555973), and Streptavidin‐FITC (BD, San Jose, CA, #554060). Flow cytometry was conducted on a Stratadigm S1000EXI or Beckton Dickinson (BD) dual‐laser FacsCalibur flow cytometer. Analyses were conducted using FlowJo v8.7 (FlowJo, LLC) software.