Calibration curves for MTT test Sample Clauses

Calibration curves for MTT test. Cell counting: Under sterile conditions 100 µl of cell suspension was mixed with an equal volume of Trypan blue dye by gentle pipetting. Trypan blue dye only stains dead cells. The number of viable cells was counted in a pre-cleaned haemocytometer and the concentration of viable cells was determined using the following equation: Concentration of viable cells (cells/ml) = A x B x C Where A is the mean number of viable cells counted, B is the dilution factor and C is the correction factor (104). Before performing an MTT test, calibration curves were constructed to determine the optimal cell seeding density for the experiment. The cells were seeded into a 96-xxxxx plate at concentrations of 5x105, 2.5x105, 1.25x105, 6.25x104, 3.125x103, 1.56x103, 7.81x102, 3.91x102 and 1.95x 101 cells/ml. The experiment blank consisted of 100 µl of medium only. The cells were incubated for 24hrs at 37 ºC, 5% CO2. Solutions: A 1 mg/ml MTT solution was made by dissolving 40 mg of MTT powder in 40 ml sterile PBS (the mixture was sonicated for a few seconds to aid dissolution) and stored in the fridge (2-8 C). SDS solution was prepared by dissolving 4g of SDS powder in 20 ml pure water. After sonication, 20ml of DMF was added to the SDS solution to give a final concentration of 10% SDS. After 24 hr incubation, 25 µl of filtered MTT solution (1mg/ml) mixed with 25µl medium was added to each well and the cells re-incubated for 4hrs. Normally the dissolved MTT is converted to insoluble purple formazan crystals by cleavage of the tetrazolium ring by dehydrogenase enzymes in viable cells. This water insoluble formazan is solubilized using SDS. At the end of the incubation period, the medium was removed from the plate and the converted dye was solubilized by the addition of 100 µl of SDS solution to each well (including the blank). The cultures were incubated overnight and absorbance was measured at two wavelengths; 570 and 650 nm. A calibration curve was constructed by plotting number of cells against the mean absorbance (Appendix).