Macs anti fitc microbeads

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)].

Frozen SVF suspensions were thawed and for each donor vials were split to allocate half for MACS sorting and half for plastic adherent culture. ASC were enriched using MACS™ anti-FITC microbeads (Miltenyi) according to the manufacturer’s instructions with an antibody cocktail consisting of 2.5 µl of each of the following antihuman FITC-conjugated antibodies: CD31 (clone MW59), CD45 (clone HI30), CD146a (PIH12), and CD235a (clone H1246) (all from BioLegend). 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. 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) per 10⁷ cells for 15 min at 4°C and washed with MACS buffer. These were resuspended in 500 μl of buffer and applied to a precooled LS column (Miltenyi). Post sort purity was assessed by flow cytometry using CD73, CD90, CD31, CD45, CD34, and CD146 antibodies (see Supplementary Table 2).

Epididymal adipose tissue fat pads were minced and stromal Vascular Fraction (SVF) separated by collagenase (2 mg mL^–1) digestion. SVF cells were incubated with a Fluorescein isothiocyanate (FITC)‐conjugated F4/80 primary antibody (BioRad catalog no.: MCA497F) at 4 °C for 30 min. Cells were washed, centrifuged, and incubated with MACS anti‐FITC microbeads (Miltenyibiotec catalog no.: 130‐048‐701) for a further 15 min and were separated using a MACS LS column according to manufacturer's instructions. F4/80⁺ cells (7 × 10⁵ cells per well) were incubated with ³H‐cholesterol (1μCi mL^–1) overnight and equilibrated in 0.2% BSA containing media for a further 18h. Percentage ³H‐cholesterol efflux to ApoA1 (20 μg mL^–1; an indirect measure of ABCA1‐dependent efflux) and serum (2.5%; a measure of total efflux via all transporters) over 4 h was determined. Cellular protein was harvested in RIPA buffer for immunoblot analysis.