Dmem f12

After transfection with 1.0 µg SBE-pNL1.2 per 100,000 cells, cells were detached by trypsinization and seeded in white polystyrene 96-well plates at a density of 3 × 10⁴ cells/cm² for the SW1353 and 8 × 10⁴ cells/cm² for the G6 and H11 chondrocytes. Cells were serum-starved overnight, 1 h pre-incubated with DMEM/F12 (control), rhIL-1β (R&D Systems, Minneapolis, MN, USA) or OAS-cm and then stimulated with rhTGF-β1 (Biolegend, San Diego, CA, USA) for 5 h. Cells were lysed 5 h post-stimulation using 30 µL ultra-pure water. An equal amount of Nano-Glo luciferase reagent (Promega, Madison, WI, USA) was added and luminescence was determined at 470–480 nm (Clariostar, BMG Labtech, Ortenberg, Germany). Each condition was performed in quadruple and the mean per experiment was depicted.

Sphere-forming assays in methylcellulose-based medium were performed as previously described (34) with some modifications. Briefly, medium containing 0.8% methylcellulose was used instead of liquid medium, and other conditions were the same as in liquid medium. Three percent methylcellulose was purchased from R&D Systems (Minneapolis, USA), and a stock solution was made of 2% methylcellulose in DMEM/F12. A final concentration of 0.8% methylcellulose in DMEM/F12 was used for cell culture. Approximately 2 × 10⁴ cells from disaggregated CTSC spheres were resuspended in a semisolid medium and plated in 6-well plates. After 7 days, the spheres were microscopically visualised, and the diameters were measured.
Serial passage experiments were conducted as described previously (18) with some modifications. Briefly, 5,000 cells from disaggregated CTSC spheres were plated on 150-mm poly-HEMA-treated cell culture plates. After 10 days, the spheres were disaggregated and re-plated at the same density. The sphere-forming efficiency (SFE) at each passage was obtained by calculating the percentage of the number of spheres divided by the number of cells plated.

The human SSC line was established by overexpressing human SV40 large T antigen in human primary GPR125-positive undifferentiated spermatogonia in our lab (Hou et al., 2015 (link)), the cell line was positive for a series of SSC markers including GPR125, GFRA1, PLZF, UCHL1, and THY1, and it can be expanded in vitro for a long time.
The human SSC line cells were cultured in DMEM/F12 (Gibco, Grand Island, NY, United States) with 10% FBS (Gibco) and 100 unit/mL streptomycin/penicillin (Invitrogen, CA, United States) at 34°C in 5% CO₂ incubator. The cells were passaged every 4 days using 0.05% trypsin and 0.53 mM EDTA (Invitrogen). In order to seek which growth factor mediates TCF3, the cells were incubated in the DMEM/F12 with the addition of 10 ng/mL GDNF (R&D Systems, MN, United States), 10 ng/mL FGF2 (R&D Systems), 10 ng/mL epidermal growth factor (EGF) (Sigma, MO, United States), or 1,000 IU/mL LIF (Cyagen, Suzhou, China).

Cells were dissociated and cultured in the modified DMEM/F-12 supplemented with N2 (R&D Minneapolis, MN, USA), 10 ng/mL epidermal growth factor (Invitrogen, Carlsbad, CA, USA), 10 ng/mL basic fibroblast growth factor (Invitrogen) and penicillin/streptomycin at 10⁴ live cells/low-attachment six-well plate (Corning Inc., Corning, NY, USA) along with various concentration of berberine. Cell density/ 10,000 cells were calculated and shown as percentage of control group.

Osteogenic differentiation and adipogenic differentiation were evaluated by growing hAMSCs for 14 days in αMEM with 10% FBS supplemented with osteogenic and adipogenic supplements, respectively (R&D Systems, Minneapolis, MN, USA). For chondrogenic differentiation, we used DMEM/F12 containing both ITS and chondrogenic supplement (R&D Systems, Minneapolis, MN, USA). We used a specific antibody panel consisting of anti-hFABP4 (adipocyte marker), anti-hOC (osteocyte marker), and anti-hACAN (chondrocyte marker) (R&D Systems, Minneapolis, MN, USA) to analyze the respective mature phenotypes by immunofluorescence. Samples were analyzed using an EVOS™ FL Digital Inverted Fluorescence Microscope (Thermo Fisher Scientific, Waltham, MA, USA).