Mtt cell assay kit

The viability of SHEDs cultivated in various eluates was examined through MTT cell assay kit after 24 h, 48 h, and 72 h of incubation (EZ count, HiMedia, India). SHEDs cultured in DMEM and 1 mM hydrogen peroxide were used as positive and negative control specimens, according to ISO 10993 standards.[10 ] MTT was added to all of the wells in question and cultured for 240 min before the procedure was stopped with the addition of dimethyl sulfoxide. A microplate reader (BioTek Instrument, USA) was used to determine AB_{570 nm} with a reference wavelength at AB₆₃₀.[13 (link)] The cement dilution was chosen for gene expression analysis based on the results of the cell viability test.

MTT assay
The cell viability was assessed by MTT assay (21 (link)), which determines the metabolically active mitochondria of intact cells. The assay was carried out using MTT cell assay kit, following the protocols described by the manufacturer's (HiMedia, India). Briefly, PLC/PRF/5 cells were seeded in 96-well plates (Greiner, Frickenhausen, Germany) with 5×10³ cells/100 μL and incubated for 24 h at 37 °C. The cells were then treated with PEF, CHF, EAF and MeF (100 μg/ mL and 50 μg/ mL), 5-FU (50 μg/ mL and 25 μg/ mL) and DMSO (0.1% v/v) and incubated for another 24 h at 37 °C in a 5% CO₂ atmosphere. The assay was performed by the addition of premixed MTT reagent, to a final concentration of 10% of total volume, to culture wells containing various concentrations of the test substance and incubated for further 4 h. During 4 h incubation, living cells converted the tetrazolium component of the dye solution into a formazan product. 100 μL of the solubilization solution (provided with the MTT assay kit) was then added to the culture wells to solubilize the formazan product and the absorbance at 570 nm was recorded using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). The experiments were performed in triplicate. Percentage inhibition was calculated using the formula,