In order to measure the activity of a transcription factor we treat our hepatoma cells with different test compounds for 4 hours and measure the activity of a reporter gene. To see whether the test compounds affect the viability of the cells and thereby the expression of the reporter gene, we have measured LDH release from the cells, as this assay can be multiplexed with the reporter gene assay without problems. However, I want to ask whether LDH assay is a proper assay for cytotoxicity as the exposure time is short, and maybe the cell membrane integrity is not affected? Is it better using MTT assay?
Effects of compounds on cells are dependent on the specific compound and also the concentration and exposure time. I think the MTT will give you effects at earlier times than the LDH assay. Why not use XTT as alternative to MTT. Saves time as you do not have to solubilize formazan after your reaction.
I agree with Edmund that the effect of compounds on cells being dependent on the nature of the compound, dose used and exposure time. LDH assay is based on the fact that dead cells, whether they die by apoptosis, necrosis or otherwise, release LDH due to loss of plasma membrane integrity. In the case of necrosis you detect LDH early on, whereas in the case of apoptosis, membrane integrity is maintained until late stages of the process.
MTT/XTT assay is based on the mitochondrial function, which is affected earlier in both apoptosis and necrosis, than membrane permeablization. As such it is a better assay than the LDH, I think. However, some toxins/drugs affect mitochondrial enzymes may give you false loss of cell viability, though they may not be killing the cells in the 4 hour time scale. Perhaps combine it with looking at cells morphology in culture and counting number of cells rounding up (one of the early visible events during apoptosis).
Some groups often uses loss of mitochondrial membrane potential as quick flow cytometry-based assay (using TMRE) for determining toxicity of drugs before doing more elaborate and specific cell death assays.
If the compound is affecting reporter gene expression before 4 hr than it indicates that the compound enters the cell before 4 hr (mRNA turn over time is also imp if the compound is toxic nd not static). So LDH assay should work because it measures the LDH released by dead cells and damaged membrane. If you want to measure cell death, WST-1 or XTT is good, if u want to quantitate cytotoxicity of the compound on membrane, only than LDH assay should be used.
But for LDH, before giving the treatment, replace the media with fresh media (to remove the LDH released during cell seeding process).
Thank you all very much for your great answers. I agree that the cytotoxicity is dependent on conc and exposure time, however, the conditions of our reporter gene assay is already optimized to 4h of exposure. In the assay the highest concentrations of some compounds decreased the reporter gene expression, and that could have been confounded by cytotoxcicity. We will now combine MTT and LDH for some of the compounds to see whether the results are comparable.
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