Oct 22, 2024
ATP Extraction & Measurement
- 1UCAM
Protocol Citation: Mariano MP Marin-Blazquez 2024. ATP Extraction & Measurement. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9296ml3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 22, 2024
Last Modified: October 22, 2024
Protocol Integer ID: 110522
Abstract
Protocol for extracting and quantitatively measuring ATP content in cultured cells
Materials
- EDTA
- TCA
- mQ H2O
- Material needed for cell trypsinization
- PBS
- Trypsin
- DMEM medium
- 15 ml Falcon tubes
- Serological pipettes
- Ice
- ATP Determination Kit from Invitrogen
- White opaque 96-well microplates with opaque bottoms
- Microplate luminescence reader
Safety warnings
The Standard Reaction Solution must always be protected from light. After every sample is prepared,
the plate must be loaded in the dark to avoid luciferin degradation. This protocol is designed to be used with cell samples coming from at least a T-25 flask. Lower cell numbers may result in undetectable ATP amounts.
ATP Extraction
ATP Extraction
Prepare 1% (w/v) TCA by diluting 1 g of TCA for each 100 ml of mQ H2O
Trypsinize cells and resuspend in 1 mL of PBS
Add 500 µL/sample of 1% cold TCA
Vortex
Incubate for 10 min on ice
Centrifuge at 12,000 g for 10 minutes
Reagent preparation
Reagent preparation
If using the kit for the first time: Prepare a 100 mM DTT stock solution by adding 1,62 mL of H2O mQ to the bottle containing 25 mg DTT. Aliquot into ten 160 µL volumes and store frozen at ≤20 ºC. Stock solutions of DTT are stable for six months to one year. Thawed aliquots should be kept on ice or at 4 ºC until ready to use
Make 1 mL of 1X Reaction Buffer by adding 50 µL of 20X Reaction Buffer to 950 µL of H2O mQ
Make 1 mL of 10 mM of luciferin solution by adding 1 mL of 1X Reaction Buffer to one vial of luciferin (blue cap). Protect from light until use. This stock solution is reasonably stable for several weeks if stored at ≤ 20 ºC, protected from light. Divide in 250 µL aliquots
Prepare a 10 mL Standard Reaction Solution, which will be enough for one microplate as follows:
8.9 mL H2O mQ
0.5 mL 20X Reaction Buffer
0.1 mL 100mM DTT
0.5 mL 10 mM luciferin
2.5 µL luciferase
Gently invert the tube to mix, do not vortex. Keep the reaction solution protected from light until ready to use. Although the solution may be stored at 2-6 ºC protected from light for several days, assay sensitivity will diminish with time.
ATP standard curve preparation
ATP standard curve preparation
Prepare a 100 µM ATP stock solution by adding 1 µL of 5 mM ATP standard solution to 49 µL of H2O mQ (total volume: 50 µL)
Prepare the following dilutions
10 µM 10X. Add 10 µL of the stock solution to 90 µL of H2O mQ
1 µM 10X. Add 10 µL of the previous solution to 90 µL of H2O mQ
0.5 µM 10X. Add 50 µL of the previous solution to 50 µL of H2O mQ
0.25 µM 10X. Add 50 µL of the previous solution to 50 µL of H2O mQ
0.1 µM 10X. Add 40 µL of the previous solution to 60 µL of H2O mQ
0.05 µM 10X. Add 50 µL of the previous solution to 50 µL of H2O mQ
0.025 µM 10X. Add 50 µL of the previous solution to 50 µL of H2O mQ
To the microplate, add two to three duplicates of standard reaction solution as a blank measure of background luminescence
Add two to three duplicates of each 1X dilution to the microplate, adding 10 µL of the dilution and 90 µL of Standard Reaction Solution
ATP measurement
ATP measurement
Prepare several serialized dilutions for ATP determination of the sample
Add 10 µL of the serialized dilutions of the sample and 90 µL Standard Reaction Solution, taking into account that this is also a 1:10 dilution on the microplate
Incubate for 10-15 minutes at room temperature and protected from light
Measure at 560 nm, remove background signal and standardize using the Standard curve