Cell death or cytotoxicity is classically evaluated by the quantification of plasma membrane damage. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in all cells, and is rapidly released into the cell culture supernatant upon membrane damage or cell lysis. The LDH released to media reduces pyruvate to lactate by oxidizing NADH to NAD+. The amount of released LDH is estimated by the quantification of NADH consumption in the supernatant. As this assay is carried out with an aliquot of the supernatant and leaves the cells undisturbed, it is also ideal for longer kinetics testing. The assay can be performed in accordance with Good Laboratory Practices.
Typically, BALB/c 3T3 cells (clone 31) 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 to eight test compound concentrations covering a large range. After 24 h exposure, NADH consumption in the supernatant is determined for each treatment concentration and compared to that determined in control cultures. Negative controls (blank, untreated and solvent) and four concentrations of positive control (sodium lauryl sulfate or H2O2) are tested. A solution 1% Triton is used to generate the reference value for the total amount of LDH.
The quantity of NADH is measured by spectrometry at 340 nm, and is directly proportional to the number of cells with an intact membrane. A calculation of cell viability expressed as relative LDH activity in comparison to the maximum value generated by the Triton X-100 solution. 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.
The experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 4-6 weeks of the experiment start date.