Purification of Adipose Stem Cells

Frozen SVF suspensions were thawed and, for each donor, vials were split to allocate half for MACS sorting and half for plastic adherent culture. Both of the isolation methods were conducted in parallel and cells from each donor were cultured separately. ASC were enriched while using MACS™ anti-FITC microbeads (Miltenyi, Bergisch Gladbach, Germany), as described previously [3 (link)]. The antibody cocktail consisted of 2.5 µls of each of the following anti-human FITC-conjugated antibodies: CD31 (clone MW59), CD45 (clone HI30), CD146a (PIH12), and CD235a (clone H1246) (all from BioLegend, San Diego, CA, USA). In brief, the SVF single cell suspension was pelleted and resuspended in 100 μL MACS buffer per 10⁷ cells and incubated with the antibody cocktail on ice for 10 min. protected from light. The cells were washed twice with MACS buffer and resuspended in 90 μL of buffer per 10⁷ cells. Cells were then incubated with 10 μL of anti-FITC MACS microbeads (Miltenyi, Bergisch Gladbach, Germany) per 10⁷ cells for 15 min. at 4 °C and washed with MACS buffer. These were resuspended in 500 μL of buffer and then applied to a pre-cooled LS column (Miltenyi, Bergisch Gladbach, Germany). Post sort purity was assessed by flow cytometry, as described previously [3 (link)].