Viable cells depend on an intact mitochondrial respiratory chain and an intact mitochondrial membrane. Respiratory reactions depend on mitochondrial succinate dehydrogenase system that is only active in viable cells. This enzyme can cleave the yellow tetrazolium salt XTT (2,3-bis[2-methoxy-4-nitro-5-sulfopheny]-2H-tetrazolium-5-carboxyanilide inner salt) generating orange formazan. The toxicity of a test substance decreases the number of living cells resulting in lower amount of orange formazan in the sample. The assay can be performed in accordance with Good Laboratory Practices. The experiment can be designed to quantitatively evaluate medical devices according to the current ISO guideline – 10993-5, “Test for in vitro cytotoxicity” (2009) or to evaluate chemical compounds.
Typically, L929 cells are seeded into 96-well plates and maintained in culture for 24 h (~ 1 doubling period) to form a semi-confluent monolayer. Cells are then exposed eight test compound concentrations covering a large range. After 24 h exposure, formazan formation is determined for each treatment concentration and compared to that determined in control cultures. Negative controls (blank, untreated and solvent/ZBDC) and four concentrations of positive control (sodium lauryl sulfate, H2O2 or ZDEC) are tested.
The quantity of formazan is measured by spectrometry at 450 nm, and is directly proportional to the number of cells with an intact respiratory chain. A calculation of cell viability expressed as formazan is made for each concentration of the test substance by using the mean formazan of the replicate values per test concentration. This value is compared with the mean formazan of the blank value. Relative cell viability is then expressed as a percentage of untreated. The inhibition of growth percentage is calculated. If the test substance induce a cytotoxic effect on the cells, the IC50 (i.e. the concentration producing 50 % reduction of viability) is calculated from the concentration-response. If achievable, the eight concentrations of each compound tested should span the range of no effect up to total inhibition of cell viability. If the relative cell viability for the highest concentration of the sample is = 70 % of the control group, then the material is considered non-cytotoxic.
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Experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 3 weeks after the experiment start date.