I'm conducting tests to measure free radical formation (using H2DCFDA), following exposure of my cultures to various toxic compounds. Should I normalise the readings I obtain to those I got from MTT tests to measure cell viability of these cultures?
DCF is a fluorescence readout (by default, the fluoresence increase is nonlinear relative to substrate concentration) while MTT is a colorimetric assay based on absorbance (a measure of NADPH flux), which does not "exaggerate" the effect size as much as fluoresence does.
Since ROS is a mixture of different reactive oxygen species with different chemical properties and formation dynamics, it may not be very informative to try to find a correlation between ROS and a single metabolite, NAD(P)H.
In short, it is better to show the data side by side, without making claims of direct correlation (which requires statistical proof). They are just collateral events, and it is a safer interpretation.
It is better to do correlation between both reading in each parameter.
Thank you, could you please explain this better to me? What do you mean by correlating both readings in each parameter? Thanks again!
I wouldn't trust normalising data to MTT as it is not necessarily linked to cell number. Better to use A DNA stain ie. Hoescht or propridium iodine using a freeze thaw protocol. This data could be collected on the same plate as the ROS assays or collected from a parallel plate. See Toxicology. 2015 Mar 5;331:47-56.
I can send over the PI freeze thaw plate based protocol if you let me know your E-mail address.
We find this approach better than Hoescht as there are no wash steps before reading. This way you don't loose the sick but still viable cells.
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