Dimethyl sulfoxide (dmso)

For the cell viability assay, 100 μM S100A9 in the presence and absence of 5 μM of lipidated and lipid-free ApoE isoforms were aggregated at 42 °C for 72 h without agitation. For the control of the native state, ApoE and S100A9, both purified by gel filtration, were mixed in the above concentration just before adding to the cells. Cells were seeded at 80,000 cells/well in flat base 96-well cell culture plates (Sarstedt, Nümbrecht, Germany) coated with 50 µg/mL poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) in cell culture medium. After 24 h, the cells were washed twice with PBS before the addition of 100 µL reduced serum Opti-MEM medium per well (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 10 µL S100A9/ApoE isoform mixture, both native as well as aggregates, was added to each well, and the cells were incubated for a further 24 h. Thiazolyl blue tetrazolium bromide (MTT, M2128, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile water at a concentration of 5 mg/mL and 11 µL was added per well. Cells were incubated in the dark at 37 ℃ in a 5% CO₂ incubator for 6 h to allow for the formation of purple-black formazan crystals. The medium was then replaced with dimethyl sulfoxide (VWR) and absorbance was recorded at 570 nm using a BioTek Synergy 2 plate reader (Agilent, Santa Clara, CA, USA).

The cell viability assay was conducted using 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT; Sigma Aldrich, St. Louis, MO, USA). NPC-039 and NPC-BM cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells per well and exposed to the indicated dose of picrasidine I (0, 10, 20, 40 µM) for 24, 48, or 72 h, respectively. The cells in the vehicle group (0 µM) were treated with 0.1% dimethyl sulfoxide (DMSO; Sigma Aldrich). Subsequently, MTT was added to each well and incubated with cells for 3 h at 37 °C. Formazan accumulated in the wells was dissolved with DMSO and measured at a wavelength of 570 nm using a microplate reader (BioTek, Winooski, VT, USA). In a further experiment, cells were transfected with human small-interfering ribonucleic acids (siRNAs) for HO-1 and scrambled siRNA, prior to picrasidine I (40 µM) treatment.

In order to test the direct toxicity of silicone oil on the microglia cells, an MTT assay (3-(4,5-dimethylthiazol-2-yl)2,5diphenyltetrazoliumbromid) was conducted [21 ]. Cells were incubated with different concentrations of emulsified silicone oil for 24, 48, or 72 h and incubated with MTT (Sigma-Aldrich, Steinheim, Germany) in DMEM (without phenol red) for 2 h. Supernatant was discarded, and DMSO (Roth) was added to the cells and rocked. The extinction of the supernatant was measured at a wavelength of 550 nm (ELx800, BioTek, Bad Friedrichshall, Germany), and DMSO served as a blank control. Untreated cells served as control and were set at 100%.

The effects of either HG and/or GRIM-19 over-expression on HeLa and H9C2 cells viability were determined by the nicotinamide,3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye uptake method. Briefly, the cells were equally distributed in 96-well plates at a density of 1 x 10⁴ cells/well (counted by a hemocytometer). Cells were treated with different glucose level for 48 hours. Before incubation with MTT, DMEM (serum10%) medium was removed and the final concentration of MTT (Sigma, St. Louis, MO, USA) at 5 mg/ml in DMEM medium and incubation was for 6 h in the dark at 37°C. Then, the supernatant was removed from each well. The colored formazan crystal produced from MTT was dissolved in 150 μL DMSO and cell viability was determined spectrophotometrically at 490 nm (BioTek, Vermont, USA).

A total of 1 × 10³ cells (in 100 μl) was seeded into a 96‐well plate and allowed to grow for the indicated time period before analysis. Thereafter, 5 mg/mL MTT solution was added to individual wells and the 96‐well plate were incubated in a 5% CO₂ incubator at 37°C for 4 h. Then removed the medium, 100 μl dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals and the absorbance at 595 nm wavelength was measured (Bio‐Tek, USA).