Penicillin streptomycin amphotericin b suspension 100

HepG2 cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin–amphotericin B suspension (×100) (Wako, Japan) in an incubator (37 °C, 5% CO₂). The cultured cells were inoculated in 96-well plates at 5000 cells per well and cultured overnight. Then, the cells were treated with 1 mM free fatty acids (FFAs) for 24 h to establish a fatty liver cell model. HepG2 cells with sufficient lipid accumulation were treated with three different types of NBW (20%, 40%, 60%, 80%, 90%), cordyceps extract (0.1, 0.2, 0.4, 0.8 mg/mL) and Wi-A (0.1, 0.2, 0.4, 0.8 µM) at different concentrations for 48 h, respectively. Subsequently, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was added to the medium (5 mg/mL). After incubation for 4 h, the medium in each well was removed and 100 µL DMSO was added into each well. The absorbance of the solution was measured at 450 nm.

Primary ADSCs were isolated from 3- to 4-week-old female CGE transgenic mice. Mouse subcutaneous fat pads were dissected, washed with phosphate-buffered saline (PBS), diced, and dispersed in 2 mg/mL type I collagenase at 37 °C for 30 min. Dulbecco’s modified Eagle medium containing 10% fetal calf serum was then added to stop the enzyme reaction, and undispersed tissue was removed using a cell strainer. The cell suspension was centrifuged at 440× g for 5 min, and the supernatant was removed. Cells were washed with PBS, and supernatant removal via centrifugation, repeated twice. Finally, cell pellets were dispersed with a MesenCult Expansion Kit (Mouse) (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with penicillin–streptomycin–amphotericin B suspension (100×) (FUJIFILM Wako Chemicals, Osaka, Japan), plated on cell culture dishes, and incubated at 37 °C in a humidified incubator with 5% CO₂. Cell suspensions of approximately 1 g of fat pads from five female mice were plated on a 10 cm culture dish.

Styrene was purchased from Chuo Kasei Co., Ltd (Osaka, Japan). Acrylic acid solution (80%) was purchased from Toagosei Co., Ltd (Tokyo, Japan). Itaconic acid was purchased from Fuso Chemical Co., Ltd (Osaka, Japan). Sodium dodecylbenzene sulfonate was purchased from Kao Co. (Tokyo, Japan). Potassium persulfate was purchased from ADEKA Co. (Tokyo, Japan). IR-1061 and pluronic F127 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), Dulbecco's phosphate buffered saline (D-PBS), D-MEM (high glucose) with l-glutamine and phenol red, and penicillin–streptomycin–amphotericin B suspension (×100) (antibiotic–antimycotic solution) were purchased from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Methoxy PEG modified with oligoamine at one end, Blockmaster™ CE210, was purchased from JSR Life Sciences Co. (Tokyo, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and lactate dehydrogenase (LDH) assay kit-WST were purchased from Dojindo Laboratories (Kumamoto, Japan). The mouse fibroblast cell line NIH-3T3 was purchased from ATCC (Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Cytiva (Mariborough, MA, USA). All the reagents were used without further purification.