Celltiter 96 aqueous non radioactive cell proliferation assay mts

SH-SY5Y cells were seeded in 96-well culture plates at a density of 12 × 10⁴ cells per well in DMEM/F12 (1:1) medium supplemented with 10% fetal bovine serum, 1× non-essential amino acids, 100 units/mL penicillin and 10 mg/mL streptomycin (Dutscher, 67170 Brumath, France). After 48 h of incubation, the cultures were treated with 100 µL of the test compounds or DMSO (0.1%) in the same medium. Following 24 h, the cells were co-incubated with H₂O₂ (200 µM) or a mixture of oligomycin/rotenone (10 µM/ 30 µM) with or without the tested compounds at noncytotoxic concentrations for an additional period of 24 h. The percent of cell viability was measured using CellTiter 96 AQueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Charbonnières-les-Bains, France).

Cell viability was measured using the CellTiter 96® Aqueous Non-radioactive Cell Proliferation MTS Assay (Promega, Madison, WI). The MTS assay is based on the bio-reduction of a tetrazolium compound (MTS) to a soluble formazan product by metabolically active cells. The amount of formazan product formed is directly proportional to the number of live cells, thus providing a quantitative measure of cell viability. After TEER measurement, 200 μL fresh Maintenance Medium containing the MTS/PMS reagent were added to the apical compartment of the culture inserts and incubated for 1 h at 37°C to allow the metabolic conversion of tetrazolium to formazan. One hundred and twenty μL of the apical medium then were transferred to a clear 96-well plate. The optical density of the apical medium was measured at 490 nm using a Synergy H4 plate reader (BioTek, Winooski, VT). Triplicate cultures were used for each treatment concentration and time point.

BV2 cells were plated in 96-well plates at 5 × 10³ cells/well. After 24 h of cell plating, media was replaced by fresh RPMI/ITS containing 100 ng/mL LPS, or no addition, and cells were incubated for additional 24 h. Then, BV2 cells were exposed to 25 µM zVAD-fmk for additional 24 h. Nec-1 (30 µM) was added 1 h before zVAD-fmk. Cellular metabolic activity was measured using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Madison, WI, USA). Changes in absorbance were measured at 490 nm using GloMax® Multi Detection System (Sunnyvale, CA, USA).

Normal skin fibroblasts were cultured in DMEM/F12 (Dulbecco's modified essential medium/Ham's 12 nutrient mixture, Gibco). Colorectal cancer cell lines, Caco‐2 and HCT‐116, were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium). Both media were enriched with 10% Fetal Bovine Serum, 100 U/ml of Penicillin, and 100 µg/ml of Streptomycin. Cells were maintained at 37ºC in a humidified 5% CO₂ incubator. Cell viability was determined by vital staining with trypan blue (0.4% (w/v); Sigma), and cells were counted using a light microscope (Bardaweel et al., 2015).
In vitro assessment of the oil antiproliferative activity was carried out using the Promega CellTiter 96^® AQueous Non‐Radioactive Cell Proliferation (MTS) assay to evaluate the number of viable cells in media (Promega 2005). The oil was applied to test wells at concentrations of 5, 10, 20, and 50 mg/ml and incubated at 37ºC with 5% CO₂ for exposure period of 48 hr. After the completion of the incubation time, the MTS mixture (20 μl/well) was employed. A microplate enzyme‐linked immuno‐assay (ELISA) reader was utilized to read absorbance of the formazan product at 492 nm. Each point was performed in triplicates (Bardaweel et al., 2015).

CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Madison, WI) with minor modification to the manufacturer's instructions. Transfected primary limbal cells were seeded in triplicate at 40,000–60,000 cells/well in BM protein coated 48 well plates. Absorbance was read on day 3 and day 7 post-transfection using the DTX 880 (Beckman Coulter, Brea, CA) at 450 nm following 2-hour incubation at 37°C and 5% CO₂ with MTS reagent. Data were collected with Soft Max Pro 6.3 (Molecular Devices, Sunnyvale, CA). Average numbers of viable cells were calculated using linear regression generated from a standard curve possessing an R-squared value of 0.985 or greater.

Proliferation of splenocytes was measured by CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation (MTS) assay according to the manufacturer’s instructions (Promega). Briefly, rabbit and mouse spleen cells (1×10⁵/well) were treated with 2µM of CpG-ODN for 48h. MTS/PMS solutions were added into each well. After 2h the absorbencies at 490nm were measured by an Envision Alpha Multilabel Reader (PerkinElmer).

Cells were treated for different times in 96-well culture plates in 200 μL of culture medium. Each concentration was tested in duplicate at least thrice separately. The viable cells were determined using the CellTiter 96 AQueous non-radioactive cell proliferation MTS assay (Promega) and the inhibition rates were analysed using GraphPad 5. Cell apoptosis was assessed by Annexin-V/PI staining using a FACSCalibur flow cytometry system.