Promokine

Stably transfected PC12 cells were seeded at 10,000 cells/well into collagen-1-coated 96-well plates in 100 µl supplemented DMEM media. The following day, cells were treated with sodium succinate (Sigma-Aldrich) at 0.5, 1, 2, 10, or 20 mM in supplemented media or supplemented media alone as control. Media pH was unaffected by succinate at the indicated concentrations. Cell viability was measured after 24 h and 48 h using an XTT-based cell proliferation kit (PromoKine, PromoCell, Heidelberg, Germany). Signal was detected with a microplate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany) at 450 nm and 630 nm 4 h after addition of 25 µl reaction solution per well.
Candidate SUCNR1 inhibitors were kindly provided by Prof. Guang-Bo Ge (Table 1). Cells were treated with the inhibitors for 48 h in the presence or absence of 10 mM succinate, after which cell viability was determined.

3D co-cultures were allowed to proliferate for 7 days before myogenic differentiation was induced for 0, 14, and 28 days. Water-soluble tetrazolium salt (Wst)-8-assay (Promokine, Promocell GmbH, Heidelberg, Germany) of the seeded scaffolds was performed as triplicates as described previously [12 (link)]. For assessment of cell viability, absorbance was measured at 450 nm with Photometer Thermo Scientific™ Multiskan™ GO. Afterwards, live dead assay (apoptotoc/necrotic/healthy cells detection kit, Promokine, Heidelberg, Germany) was performed according to the manufacturer’s instructions. 10× magnified images of every quadrant of each scaffold were taken with a fluorescence microscope (IX83, cellSens, software, Olympus). ImageJ 1.53e (National Institutes of Health, Bethesda, MD, USA) was used to quantify live, apoptotic, and necrotic cells by splitting color channels and inverting blue (live cells), red (necrotic cells), and green (apoptotic cells). An automatic cell count was carried out of each channel as described above. Necrotic index (NI) and apoptotic index (AI) were calculated as number of red or green cells in relation to total cell number, respectively.

Plasma 25(OH)D concentrations were measured using an ELISA kit (Promokine, Promocell). IL-6 cytokine was quantified with Ready-SET-Go! ELISA (Life Technologies, Villebon-sur-Yvette, France). CCL5 chemokine was quantified with Instant ELISA kit (Life Technologies, Villebon-sur-Yvette, France).