Infinite f200 microplate reader instrument

The 3T3-L1 preadipocytes were induced to differentiate into adipocytes as described previously [15 ]. Briefly, the 3T3-L1 preadipocytes were seeded at 1 × 10⁴ cells/well in 48-well plates and proliferated to confluence. Adipogenesis was triggered with differentiation induction medium containing 0.5 mM 3-isobutyl-1-methylxanthine, 1.0 μM dexamethasone and 10 μg/mL insulin in DMEM with 10 % FBS. Three days later, cells were maintained in a differentiation maintenance medium containing 10 μg/mL insulin in DMEM with 10 % FBS for another 3 days. Next, the medium was replaced with DMEM containing 10 % FBS every 3 days. After 9–11 days’ incubation, more than 80 % cells were differentiated into adipocytes. Cells were treated with different concentrations of XKA extracts throughout the entire differentiation process. The 3T3-L1 adipocytes were fixed with 0.4 % paraformaldehyde (PFA) for 1 h. Then the cells were stained with Oil Red O solution for 0.5 h. The cells were washed with 70 % ethanol/H₂O (v/v) twice. Appearance was recorded by an inverted fluorescence microscope Leica DMI 6000 B (Leica Microsystems, Wetzlar, Germany). Then, Oil Red O dye in lipid droplets was eluted into isopropanol. Finally, absorbance was measured at 492 nm using an infinite F200 microplate reader instrument (Tecan Group Ltd., Switzerland) (n = 3).

Cell viability was determined by MTT assay. The 3T3-L1 preadipocytes were seeded at 3 × 10³ cells/well in 96-well plates and incubated at 37 °C. After 2-day incubation, the cells were treated with different concentrations of XKA extracts or vehicle for 48 h. At the end, the medium was removed, and 100 μL of MTT solution (0.5 mg/mL) was added to the 3T3-L1 cells. After the wells were incubated for another 4 h at 37 °C, the medium was removed, and the synthesized formazan crystals were dissolved in 100 μL of DMSO. Finally, absorbance was measured at 580 nm using an infinite F200 microplate reader instrument (Tecan Group Ltd., Switzerland) (n = 3). Data was calculated as a percentage of MTT compared to control cells.