Mtt solution

Cell viability assay was tested through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously described [25 (link)]. Briefly 1 × 10⁴ cells were seeded in 96-well plate and incubated for 24 h at 37 °C and 5% CO₂. Cells were incubated with different concertation of BPHs (50, 10, 5, 1.5, 1, 0.5, 0.1, 0.05, and 0.01 mg/mL) for 24 h. Then, cells were incubated with MTT solution (0.5 mg/mL, Invitrogen,) for 2 h at 37 °C. Subsequently, MTT solution was removed, and 200 μL of DMSO was added, and absorbance was recorded at 540 nm with a microplate reader (Multiskan, Thermo Fischer Scientific) with Skanlt Software 6.0.2 (Thermo Fisher Scientific, Italy). To test the cytoprotective effect of BPHs in the presence of H₂O₂, cells were treated in 96-well plate with the noncytotoxic concentrations of BPHs for 2 h; then, H₂O₂ 1 mM was added, and cells were incubated at 37 °C for 24 h. After incubation period, MTT assay was performed as described above. The results were determined in percentages of the viable cells compared to the untreated control.

The cells seeded onto the surface-modified PLA particles were compared using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay as described previously [72 (link),73 (link)]. Briefly, the cells cultured on the surface-modified PLA particles in each well of 24-well culture plates were rinsed with PBS (×3), then incubated in 200 µL MTT solution (5 mg/mL, Life Technologies Corporation, Eugnene, OR, USA) at 37 °C for 3 h. After careful removal of the MTT solution, 200 μL dimethyl sulfoxide (DMSO, Fisher Scientific Inc., Loughborough, UK) solution was added into each well to dissolve the formazan crystals formed in the viable cells. Aliquots of 50 μL DMSO solution from each well were then transferred into 96-well plate for optical density measurement at 570 nm (OD₅₇₀) using a microplate spectrophotometer (BioTek instruments Ltd., Winooski, VT, USA).

Cell cytotoxicity was measured using the3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT) assay. L929 cells (5 × 10⁴ cells/well) and Caco2 cells (10⁵ cells/well) were plated in 96-well tissue culture plate in complete medium (100 μL/well). The multiwell plates were incubated at 37°C, 5% CO₂ for 24 h. After 24 h, the culture medium was removed and equal volumes (100 μL) of the treatments were added to each well. In control wells, 100 μL DMEM were added. Control wells consisted of untreated cell cultures. Twenty-four hours later, proliferative cells were detected by MTT assay, according to the ISO 10993-5 International Standard procedure (43 ). The main purpose of the ISO 10993-5 procedure is to define a scheme for testing in vitro cytotoxicity of different extracts according to a multi-step approach. Briefly, cells were incubated with MTT solution (1 mg/mL, Life Technologies) at 37°C for 2 h. Then, MTT solution was removed and cells were solubilized with 100 μl of isopropanol. The formazan dye formation was evaluated by scanning multiwell spectrophotometer at 540 nm. The results were expressed as percentage of viable cells compared to controls.

In the cell proliferation assay, 1.5 × 10⁶ U2OS cells were seeded in 10 cm dishes and treated with siControl or siRB for 48 h. Cells were then collected. Furthermore, 1 × 10⁵ cells were seeded into a 24-well plate. After 24 h, cells were treated with different dosages of etoposide and olaparib for three days. Cells were then incubated with 5 mg/mL MTT solution (Life technologies, Waltham, MA, USA) in 500 μL DMEM medium at room temperature for 3 h. MTT solution was then removed, and 300 μL isopropanol was added into each well. An amount of 200 μL of the mixture was transferred to a 96-well plate. Fluorescence signals were measured by a plate reader at 570 nm wavelength. To study the cell proliferation of primary retinoblastoma cells, cells were treated with etoposide and olaparib for three days, and cell proliferation was measured by MTT following the same protocol.

Cell viability was analyzed by MTT colorimetric assay. Briefly, 20 μL 2.5 mg/mL MTT solution (Invitrogen; Cat. no. M6494) was added into the wells of 96-well plates, and THP-1-derived macrophages were incubated with MTT solution for 4 h at 37 °C. After that, cell supernatant was discarded and the formazan product was dissolved through adding 150 μL dimethyl sulfoxide (DMSO). The optical density (OD) value (570 nm) was measured using a microplate reader (Bio-Rad, Shanghai, China).

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to analyze proliferation of NB cells. After adjusting cell density to 5 × 10⁴/ml, 100 μl of SK-N-BE(2)C and SK-N-SH cells were seeded into 96-well plates and cultured for 0, 24, 48 or 72 h. Each group was prepared in triplicate. At the end points, cells were interacted with 0.5 mg/ml MTT solution (Thermo Fisher) for another 4 h, and then medium was replaced with 100 μl of dimethylsulfoxide (DMSO, Thermo Fisher) to dissolve formazan. The absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

The cell viability of RAW264.7 cells after stimulation was determined by nuclear staining with Hoechst 33342 dye (Thermo Fisher Scientific) at 10 µg/mL (in phosphate buffer solution: PBS) with 15 min incubation at 37 °C in the dark before washing with PBS and photographed by an IX81 inverted microscope (Olympus, Tokyo, Japan). The viable cells (blue colored dots) were counted against area determination using ImageJ (NIH, Bethesda, MD, USA). In addition, the metabolic activity of cells after stimulation was analyzed by the capability of reducing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay). The cells were incubated with 0.5 mg/mL of MTT solution (Thermo Fisher Scientific) for 2 h at 37 °C in the dark. Following removal of the MTT from the wells, the MTT was diluted with DMSO then wrapped to avoid light during shaking for 5 min. Finally, the reduction of the MTT was measured using a Varioskan Flash microplate reader (Thermo Fisher Scientific) at absorbance 570 nm.