I am currently trying to develop an ATP concentration measurement protocol for my human cultured cells. However, I have found little to no detailed protocol for ATP extraction using TCA (concentration, times, etc.). Could you help me establish the experimental parameters for this procedure?
Thank you in advance.
Dear Mariano,
The procedure is quite simple. You need to homogenize the cells.
You can put the acid directly on the cell pellet and immediately homogenize the cells with ultrasound, a Potter, or a Dounce. Alternatively, you can homogenize the cells and instantly add the acid. To make the procedure as rapid as possible, I use ultrasound and add the acid to the homogenate, followed by vortexing and immediate freezing.
In practical terms, for a pellet of three million cells, I add 300 µL of buffer to make the homogenate. Then, I add 20% acid (2% TCA) for one homogenate volume. For example, for 100 µL of homogenate, I count 20 µL of 2% TCA. I immediately vortex and centrifuge in a microcentrifuge at 10000 rpm and freeze at -20°C. ATP is unstable in acidic media. Therefore, the ATP assay must be carried out quickly after preparing the defecate.
To avoid this instability, we could neutralize the sample. But this hampers the luminol assay I use, for example.
I hope this answer is helpful.
Good luck with your research.
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