Glycerol kit

The s.c. adipocytes (day 9 after differentiation medium stimulation) were washed once with PBS gently and then incubated with DMEM (no phenol red and FBS) (Wisent Biotechnology, Nanjing, China) containing with or without MCM for 24 h. The culture medium of samples were collected and glycerol levels in the media were assayed using a glycerol kit (Applygen Technologies Inc., Beijing, China) according to the manufacturer’ instructions.

Mature conidia and mycelia incubated for 24 h were stained with 2.5 mg/mL of Nile Red (Macklin) to localize lipid droplets and observed under the laser confocal fluorescence microscope (Leica TCS SP8, Berlin, Germany). Fluorescence signals were detected with 514 nm excitation light, and the emission wavelength was set between 539 and 749 nm. Mycelia or conidia were observed with the objective of HC PL APO CS2 63×/1.4 OIL.
To determine the glycerol concentration, 8 mycelial discs were inoculated into 100 mL of liquid CM medium containing 0.03 g/L hemin and cultured in a shaker (175 rpm, 25 °C) for 3 d. Then, the mycelia were collected using a 200-mesh nylon net, washed with sterilized water, and the excess water was removed. Glycerol determination was measured using the glycerol kit (Applygen Technologies Inc., Beijing, China). Fresh mycelia were pulverized to powder with liquid nitrogen and incubated in the lysis buffer. Absorbance was detected using a SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA). A standard curve (r = 0.9999) was generated according to the kit instructions. All procedures were performed according to the instructions. There were three replicates for each treatment, and the experiment was repeated three times independently.

A total of 5 × 10³ cells were seeded into a 96-well plate and treated for 48 h after an overnight standing (the cells were adhered). The medium was collected and centrifuged at 12,000×g for 5 min at 4 °C prior to determination. Glycerol in the supernatant was calculated using colorimetric assay according to the manufacturer’s instructions (Glycerol Kit, Applygen, Beijing, China).