Fluorobrite dulbecco s modified eagle medium dmem

At the scheduled time points, vitrified samples were eluted using FluoroBrite Dulbecco’s Modified Eagle medium (DMEM) (Thermo Fisher Scientific, CA) with gently vortexing and a 10-min incubation at the room temperature. The eluted mRNA (125 ng) was loaded and run on a 1.2% agarose gel, stained using SYBR™ Green II RNA gel stain (Thermo Fisher Scientific, CA), visualized under Gel Doc EZ System (BioRad, CA), the images were captured, and the percentage of band intensity calculated with respect to a control consisting of mRNA stored frozen samples using the Image Lab 6.0.1 software (BioRad, CA). A Millennium™ RNA marker size range from 0.5 to 9 kb was used as a ladder.

The wild-type OP9 cells and the dual-readout OP9 cells were maintained according to published protocols (7 (link)). Briefly, the cells were cultured in full growth medium consisting of minimal essential medium-α (MEM-α) (ThermoFisher Scientific) containing 1 unit/mL penicillin, 1 mg/mL streptomycin, and 292 μg/mL l-glutamate supplemented with 20% fetal bovine serum (FBS). To induce differentiation, 100 nM rosiglitazone (Cayman) or the adipogenic mixture (DMI), consisting of dexamethasone (1 μM; Sigma-Aldrich), IBMX (250 μM; Sigma-Aldrich), and insulin (1.75 nM; Sigma-Aldrich), was used. For live-imaging experiments, the differentiation stimuli were added to Fluorobrite Dulbecco’s modified Eagle medium (DMEM) (ThermoFisher Scientific), supplemented with 10% FBS, and then cells were continually imaged for 4 d. The small molecule LH846 (Cayman) was used at a concentration of 4 μM. For fixed-cell experiments, stimuli were added to MEM-α (ThermoFisher Scientific) supplemented with 10% FBS for 2 d and then removed and replaced with fresh medium containing 1.75 nM insulin (Sigma-Aldrich) and 10% FBS for another 2 d.