In our experiment we are trying to assess the effect of certain drugs on ATP output. We are using an ATP Luciferase kit as a functional assay for ATP production, however the process involves lysing our cell cultures. It would really help sharpen up our measurements if I could normalise the ATP output with a reasonably accurate estimation of cell number from this lysate, either from protein, DNA or another type of quantification method, has anyone done this before?
I don't think there is a way you can get an accurate estimation of cell number from protein or DNA quantification. The only thing I can think of (to get a ballpark number) is to make a standard curve of increasing cell numbers: Plate varying number of cells in wells--- Lyse the wells and measure ATP--- see what RLU correlates with what cell number. This should tell you whether you have 3x10e5 cells, as opposed to 5x10e5 cells in your well. This approach however would not distinguish between 3x10e5 and 3.4x10e5 (as an example).
Hi Julie,
I'm afraid you misunderstand, the ATP assay cannot be used to test for cell number because it is the functional output of a drug we are treating the cells with, that is why i need some other way to estimate cell number. I was under the impression that the amount of DNA would roughly correlate to the amount of cells in a culture (obviously not exactly 1:1 because of dividing cells etc.), enough at least to give me something to normalise my ATP assay.
Pretty much, yes: you could simply run a qPCR for genomic DNA (pick a gene that isn't multicopy or profligately represented as pseudogenes, obviously, so no 18S or GAPDH).
Compare your qPCR Cq values to a standard curve, determine the number of genomes.
To be honest, even without the standard curve, the numbers are still comparable: you might not be able to tell exactly how many genomes you'd have per well, but you would still be able to demonstrate that "well 3 had 1.23x as many as well 1".
John, than you for your answer, my problem is I assume I could not just take the lysate and stick it in a qPCR reaction, i suspect I need some way to first purify the gDNA, do you know a good way to accurately and cleanly isolate the gDNA from the lysate?
Depends on the quantity. If you're using small wells and low cell numbers (which it sounds like you are) then purification methods are more likely to lose material and introduce errors than provide benefit. On the other hand, with low cell numbers the amount of inhibitory gunk in your crude lysates will probably be similarly low, so you might be able to get away with simply boiling a small portion of lystate for ~5mins to denature liberated proteases etc and then using that directly in PCR.
Alternative methods: assuming your cells grow in a nice monolayer (or, if suspension cells, are not constantly agitated), you could just take 4-5 decent phase-contrast images of randomly selected fields and do manual cell counting.
I assume you're accounting for the fact that drugs that lower ATP output will almost certainly slow cell division, yes? Are these cultures treated acutely or chronically?
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