I want to shake cells to mechanically stimulate ATP release, but am afraid that some cells will burst. Is there a good test to check the supernatant afterwards for that?
After you perform your shaking protocol, do you harvest the cells? Are you expecting the cells to come off the plate during the protocol? You could always perform a control experiment using propidium iodide and check to see if more cells stain positive after shaking. PI will only stain nuclei if the plasma membrane ruptures. If you see no increase in staining, then no significant lysis has occurred during shaking.
An even easier method is to use a viability stain, like trypan blue, which only stains ruptured cells and you can count if the number of stain-positive cells increases after shaking using a standard microscope. Again, this would be a control experiment and you wouldn't be expecting to detect ATP, but it would certainly tell you if your shaking protocol results in significant lysis.
After you perform your shaking protocol, do you harvest the cells? Are you expecting the cells to come off the plate during the protocol? You could always perform a control experiment using propidium iodide and check to see if more cells stain positive after shaking. PI will only stain nuclei if the plasma membrane ruptures. If you see no increase in staining, then no significant lysis has occurred during shaking.
An even easier method is to use a viability stain, like trypan blue, which only stains ruptured cells and you can count if the number of stain-positive cells increases after shaking using a standard microscope. Again, this would be a control experiment and you wouldn't be expecting to detect ATP, but it would certainly tell you if your shaking protocol results in significant lysis.
Leaky cells will release LDH, and Trypan Blue staining will also be good to detect loss of membrane integrity. SO, both these assays will find cells in early death....rather than fully "bursted" cells. Does ATP leak out of cells in early death? You can answer this query (provided your ATP assay is sensitive-chemiluminescence based assay should do it). Hope this helps!
Enzyme-linked immunolysis assay for tumour specific surface antigens. A. Khar and V. Sitaramam. J. Biochem. Biophys.Methods. 13, 161-169, 1986.
This paper, first to describe use of LDH in what was called enzyme linked immunolysis assay (ELILA) addresses to a statistical evaluation of this test along with chromium release and trypan blue exclusion, both of the latter performing far worse quantitatively in the statistical sense.
The question you need to answer is far more subtle. Are the cells that are releasing ATP intact? That is not as unlikely as one would tend to think.
You can actually monitor release of ATP from the cells continuously by adding ATP Reagent SL (luciferin/luciferase) to the medium measuring the light emission. Calibration is done by adding a known amount of ATP Standard. With cell suspensions this can be done in any type of luminometer. With cells growing in microplates a microplate luminometer is used. With cells growing in 35 mm Petri dishes an FB12 Luminometer can be used. If you need further information, please contact me at [email protected].
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