Poietics human adipose derived stem cell adipogenesis protocol

For assessing adipogenic capacity, in technical triplicates 30,000 SVF cells at day 34, 49, 71 and 89 of cultivation were seeded per well in a 48-well dish or transfected SGBS cells in a 12-well format were used. Cells were grown to confluence for 24 to 48 h and differentiated according to the Poietics human adipose-derived stem cell-adipogenesis protocol (Lonza) for 8 d (SVF cells) or 10 d (transfected SGBS cells).
Differentiated cells were fixed with Roti-Histofix 4% (Carl Roth), double-stained with 10 µg ml⁻¹ Nile red (Sigma-Aldrich) and 40 µg ml⁻¹ Hoechst 33342 (Sigma-Aldrich). The percentage of differentiated cells is given as mean from n = 3 technical replicates calculated as (number of counted differentiated cell per microscopic image) / (total number of cells per image). A cell was defined to be differentiated when containing at least two lipid droplets. Additionally, the cells were stained with Oil Red O (Sigma-Aldrich) solution (0.3% in 60% isopropanol) for 15 min and washed with H₂O. Oil Red O was extracted with isopropanol and absorption was measured at 540 nm.

Simpson–Golabi–Behmel syndrome (SGBS) cells were kindly supplied by Martin Wabitsch, University of Ulm, Germany [15 (link)], and were cultured in DMEM/Ham F12 medium (Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 33 μmol/L biotin and 17 μmol/L pantothenic acid. Adipocyte differentiation was performed in four technical replicates and induced by treating confluent cells with serum-free medium containing 20 nM insulin, 100 nM hydrocortisone, 0.2 nM triiodothyronine, and 0.13 nM apo-transferrin. For the first 4 days of differentiation, 25 nM dexamethasone, 500 µM isobutyl-1-methylxanthine, and 2 µM rosiglitazone were added to the medium [16 (link)].
Cell culture and adipogenic induction of primary cells from adult patients (N = 5; Lonza, Cologne, Germany) and cryopreserved aliquots of SVF cells from children included in the Leipzig AT childhood cohort (N = 7) was performed according to the Poietics™ human adipose-derived stem cell–adipogenesis protocol (Lonza). For SGBS cells and primary cells from adults, analyses were performed at different time points of adipocyte differentiation from day 0 (before adipogenic induction) until day 12. For primary SVF cells from children, measurements before (day 0) and at day 8 of adipocyte differentiation were available.