Modular multitechnology microplate reader

Cell viability was tested by the colorimetric MTT reagent assay. RAW 264.7 cells were seeded (5 × 10³ cells/each well) in a 96-well plate and cultured overnight. After pretreatment with different concentrations (0, 31.5, 63.0, 126.0, 189.0, 252.0 and 504.0 μg/mL) of 5-HMF for 6 h, the cells were stimulated with or without 1 μg/mL of LPS for 24 h. After removal of the media, 110 μL of the MTT solution (10 μL MTT plus 100 μL culture medium) was added to each well and incubated for 4 h. Then the media were removed and 100 μL of the formazan solvent was added to dissolve the purple precipitates. The absorbance was measured at a wavelength of 570 nm using a Modular multitechnology microplate reader (Thermo Fisher Scientific, Gaisburg, MD, USA). Each sample group consisted of at least three replicates.

The effect of RSV and RSV-PLGA-NPs on the proliferation of HepG2 cells before induction was measured by MTT cell proliferation and cytotoxicity assay kit (PH0533). Untreated cells were selected as controls. Briefly, HepG2 was seeded into 96-well plates at 1 × 10⁴ cells well⁻¹. RSV and RSV-PLGA-NPs were added to each well, respectively. They were incubated together with cells for 24 h. DMEM medium containing 10% FBS was used throughout. The absorbance was recorded at 570 nm by the modular multi-technology microplate reader (ThermoFisher, Varioskan LUX, USA). The effect of RSV and RSV-PLGA-NPs on cell proliferation of HepG2 cells after induction was determined by BCA protein assay kit (C503021). After the induction of OA, cells were incubated with RSV and RSV-PLGA-NPs for 24 h. The supernatant was discarded and the cells were digested. The protein content determined according to the instructions of BCA protein assay kit (C503021) could represent the cell number. The control group of RSV was treated with DMSO, and the control group of RSV-PLGA-NPs was treated with PLGA-NPs.

Cell proliferation was assessed with a CCK-8 assay (Beyotime Biotechnology, Shanghai, China), according to the manufacturer’s instructions. PASMCs were serum starved in serum-free medium for 24 h prior to the proliferation experiments. Approximately 1 × 10⁵ PASMCs were plated in 96-well plates. After different stimuli were added to the cells, they were incubated in an incubator (5% CO₂, 37 °C) for 24 h. Fresh serum-free DMEM solution containing 10 µl of CCK-8 solution was then added to each well and incubated at 37 °C for 1 h in the dark. Analyses were performed with a modular multitechnology microplate reader (Thermo Fisher Scientific) by measuring the optical density value at 450 nm.