Hello everybody. I would like to do measurements of ATP in pieces of liver from a KO mouse. I was wondering if anyone of you know a good buffer to solubilize liver pieces and then proceed with a standard luciferase assay.
I've read about using TCA 1% and EDTA 4mM but I wonder if it would be enough to lysate the liver.
Thanks in advance
Dear David,
I believe you want to measure ATP in your tissue prep. 0.5% to 2.5% TCA works well. If you want to further stabilize the extracted ATP in the tissue prep you can add ARL 67156 (Tocris, Cat. No. 1283). But TCA alone should be effective enough to degrade any ecto ATPase.
We have published some data on luminiscence ATP assay done in extracellular bathing media. We reconstituted the the ATP assay mix, containing firefly luciferase, luciferin, MgSO4, EDTA, DTT, and BSA in a Tricine buffer (Sigma, St Louis, MO).
For further reading I am sharing our published paper.
Hope that helps.
Comparing different concentrations of TCA is a good reference method. There is no Point to include EDTA as the acid form of EDTA does not bind divalent ions like magnesium and calcium. If your pieces of liver are small enough it is possible that you can use our Extractant BS (a quaternary ammonium compound), which lyses the cells and inactivates ATP degrading enzymes. All required reagents are included in the Cell Viability Kit SL. Instructions for use and an overview of optimization of ATP assays are attached.
Best regards,
Further, I strongly recommend to measure not just the [ATP] but also [ADP] and [AMP] (and maybe also [inosine]). It will add additional work but as one of my mentors likes to say, "if something is not worth doing well, it's not worth doing it at all":
Article Getting Surprising Answers to Unasked Questions
(http://dx.doi.org/10.1016/j.cell.2017.06.001).If the ratio is too different from the common (for the mouse liver) "3.5 mM/1.8 mM/0.33 mM" then the [ATP] is degraded.
This point is quite easy to miss and unfortunately this happens often:
(two excellent letters addressing this are below)
https://www.ncbi.nlm.nih.gov/pubmed/21731132
https://www.ncbi.nlm.nih.gov/pubmed/21731131
It is technically challenging to capture the ATP in the liver and in many other organs: one needs to take the liver from an anesthetized mouse and preferably clamp-freeze it in the liquid nitrogen. If mice are euthanized, then ATP degrades considerably before the liver is removed and frozen (making the results essentially useless):
http://dx.doi.org/10.1371/journal.pone.0117232
Our cell lysis buffer contains (50mM Tris, 2mM EDTA, 120mM NaCL, 1% IGEPL) with cocktail of proteinase inhibitors. Can this be used to lyse cells (monolayer culture) for optimal ATP sensitivity using ATP determination kit (A22066)?
Also, how to normalize samples, equal cell number seeding or quantify cell lysate after lysis? Thanks
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