2.4. MTT test protocol
The Caco-2, COLO 320, DLD-1‎, HCT-15‎, HCT-116‎, and HT-29 colorectal cancer cells in DMEM culture medium (Gibco, USA) with 10% FBS (Gibco, USA) and penicillin/streptomycin (100 μL/100 μg/ml) in an incubator containing 5 % Carbon dioxide with 90% humidity was stored at 37 °C.
Cancer cell lines were placed in 1640-RPMI medium from GiBco manufacturer and were cultured after adding 10% bovine serum, 1% streptomycin and penicillin antibiotics and 2% glutamine. At this stage, the cell culture flasks were kept in an incubator with 5% CO2 and 95% humidity at a temperature of 37°C, and the culture medium was replaced every three days. In this step, flasks with 80% cell density were used (flasks filled with cells up to 80% of the bottom). First, the culture medium was removed from the surface of the cells and by adding 1 ml of trypsin for 3 minutes and then adding the same volume of medium to neutralize the effect of trypsin, all the cells were separated from the flask bottom. This cell suspension was centrifuged at 1200 rpm for 4 minutes. The liquid above the sediment was discarded and 1 ml of culture medium was added to the sediment. By taking 10 µl of the cell suspension and adding the same amount of trypan blue on the surface of the neobar slide, the number of living cells was counted. The number of 10,000 cells from this cell suspension was added to each well of 96-well plates and 180 µl of culture medium was added to it. In the next step, 20 µl different concentrations of nanoparticles were added to the wells. In this research, based on the conventional concentrations of nanoparticles at the 0-1000 µg/ml, they were added to cancer cells. Another group of cells were tested as a control, without adding nanoparticles and only by adding water instead of nanoparticles, and each experiment was done in four replicates. After 24, 48 and 72 hours, the medium on the cells was replaced with a new medium. Then 20 µl MTT solution was added to each well and placed in a greenhouse for 4 hours in the dark in a CO2 incubator. During this time, the mitochondrial succinate dehydrogenase enzyme of living cells changes the yellow MTT solution into purple formazan crystals, which are insoluble in water. In the next step, 200 µl of DMSO (Dimethylsulfoxide) was added to the empty medium and shaken for 20 minutes to dissolve the light-producing crystals. In the last step, the absorbance was read with a wavelength of 492 and then 630 nm in an ELISA reader. Finally, the percentage of cell viability was calculated after dividing the optical absorbance (OD) of treated cells compared to control cells and multiplying by 100 (22,23).
The collected data were analyzed using SPSS ver22 statistical software and a one-way ANNOVA test. P <0.01 was considered remarkable.