Lipid accumulation was quantified at differentiation day 9. hMSCs differentiated in vitro were washed with PBS and fixed with 4% paraformaldehyde solution for 10 min at room temperature. Following fixation, neutral lipids were stained with Bodipy 493/503 (at 0.2 µg/mL; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, US), and nuclei (DNA) were stained with Hoechst 33342 (at 2 µg/mL; Molecular Probes) for 20 min at room temperature. Accumulation of neutral lipids and cell numbers were quantified in CellInsight™ CX5 High Content Screening (HCS) Platform (Thermo Fischer Scientific, Waltham, MA, US) with integrated “Spot detection” protocol. Total Bodipy fluorescence (lipid droplets) was normalized to the number of nuclei, representing the number of cells in each well.
The medium was collected at day 7 and 9 of differentiation of hMCSs for measuring glycerol release as an index of lipolysis, as described [27 (link)]. A standard curve ranging from 0 to 120 µM was used to calculate the concentrations of the samples. Amounts of glycerol was normalized to the number of nuclei and in each well.
The medium was collected at day 7 and 9 of differentiation of hMCSs for measuring glycerol release as an index of lipolysis, as described [27 (link)]. A standard curve ranging from 0 to 120 µM was used to calculate the concentrations of the samples. Amounts of glycerol was normalized to the number of nuclei and in each well.
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