To isolate stromal vascular fraction (SVF) cells, 10 mL each of REVOLVE ENVI–processed and decanted grafts were digested with 250 U/mL collagenase from Clostridium histolyticum (Sigma-Aldrich) at a 1:4 fat graft to collagenase solution ratio for 1 hour at 37°C with agitation. The digested suspension was then centrifuged at 200g for 10 minutes. The supernatant was removed, and the red blood cells in the pellets were lysed with ammonium-chloride-potassium lysis buffer (Fisher) for 3–5 minutes. After lysis, the remaining cells were washed with PBS and pelleted by centrifugation. The cellular pellet was resuspended in PBS, then used for SVF fluorescence-activated cell sorting (FACS) and colony-forming unit (CFU) culture analysis.
FACS analysis was performed as previously described.9 (link) Briefly, SVF cells were washed in PBS containing 0.5% (weight/volume) bovine serum albumin and stained for 30 minutes at 4°C with fluorescence probe-labeled anti-CD34, anti-CD31, and anti-CD45 antibodies (BD Biosciences, San Jose, Calif.). The CD45–/CD31–/CD34+ progenitor cell population was identified using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).
FACS analysis was performed as previously described.9 (link) Briefly, SVF cells were washed in PBS containing 0.5% (weight/volume) bovine serum albumin and stained for 30 minutes at 4°C with fluorescence probe-labeled anti-CD34, anti-CD31, and anti-CD45 antibodies (BD Biosciences, San Jose, Calif.). The CD45–/CD31–/CD34+ progenitor cell population was identified using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences).
