ATP and Lactate Quantification

Materials and Reagents

1. For intracellular ATP content evaluation
   1. Murine Embrionic Fibroblasts (MEF)
      
   2. Dulbecco’s Modified Eagle’s Medium High glucose with L-glutamine (DMEM) (Gibco^®, catalog number: 11965 )
      
   3. Foetal Calf Serum (FCS) (Gibco^®, catalog number: 16010 )
      
   4. Penicillin/Streptamycin (Gibco^®, catalog number: 15070 )
      
   5. Phosphate Buffered Saline (PBS) (EuroClone, catalog number: E0B4004L )
      
   6. Protein Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
      
   7. Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A4503 )
      
   8. NaCl
      
   9. Na₄HPO₄/NaH₂PO₄
      
   10. Glycerol
       
   11. Triton X-100
       
   12. Complete protease inhibitors (Roche Diagnostics, catlalog number: 11697498001 )
       
   13. Phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride)
       
   14. Complete protease inhibitors (Roche Diagnostics)
       
   15. ATP détermination kit (Life Technologies, catalog number: A22066 )
       
   16. Lysis buffer (see Recipes)
       
   17. Standard Reaction Solution (see Recipes)
       
       
2. For Lactate quantification
   1. MEF
      
   2. EnzyChrom^TM L-lactate Assay Kit (BioAssay Systems, catalog number: ECLC-100 )
      
   3. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Gibco^®, catalog number: 11965)
      
   4. Foetal Calf Serum (FCS) (Gibco^®, catalog number: 16010 )
      
   5. Penicillin/Streptamycin (Gibco^®, catalog number: 15070 )
      
   6. 0.4% Trypan Blue stain (Life Technologies, catalog number: T10282 )
      

Equipment

1. For intracellular ATP content evaluation
   1. 6 wells plates (Corning, Costar^®, catalog number: CLS 3506 )
      
   2. Cell Scraper (Nunc^®, catalog number: 179707 )
      
   3. Luminometer Gloomax 20/20
      
   4. Spectrophotometer
      
   5. 37 °C 5% CO₂ Cell culture incubator
      
   6. Centrifuge spinning speed: 15682,186 G-force (13,000 rpm, 8,3 cm radius)
      
      
2. For Lactate quantification
   1. 37 °C 5% CO₂ Cell culture incubator
      
   2. 6 wells plates (Corning, Costar^®, catalog number: CLS 3506)
      
   3. 96 wells plates (Corning, Costar^®, catalog number: CLS 3596 )
      
   4. Countess automated cell counter with countess Cell Counting Chamber Slides (Life Technologies)
      
   5. Plate reader at 565 nm
      

Procedure

1. For intracellular ATP content evaluation
   1. MEF are plated at 60-70% cell confluency.
      
   2. Cells are washed twice with PBS
      
   3. Cells are lysed with a cell scraper in 35 μl of lysis buffer containing phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride) for 30 min at 4 °C.
      
   4. Centrifuge at 13,000 rpm for 15 min at 4 °C.
      
   5. Supernatant is isolated.
      
   6. The sample is diluted at 1:1,000 (1 μl of sample is added to 1,000 μl of Biorad solution) in a Biorad solution (200 μl of Biorad and 800 μl of water).
      
   7. Optical Density (OD) is read at the spectrophotometer at 595 nm.
      
   8. The concentration of the sample is determined using a standard curve from BSA.
      
   9. Samples are diluted at a final concentration of 25 ng/ml.
      
   10. 250 ng of total proteins in parallel of the ATP standard curve (0 nM, 1 nM, 10 nM, 100 nM, 1,000 nM of ATP) in 10 μl is added to 90 μl the Standard Reaction Solution.
       
   11. Luciferase activity is measured with a luminometer.
       
   12.  ATP is quantified for each sample using the ATP standard curve.
       
       
2. Lactate quantification
   1. Cells are plated in 6 wells plates with 3 ml of medium (10% DMEM-FCS-1% Penicillin/Streptamycin).
      
   2. The medium is changed when the cells reach 100% cell confluence.
      
   3. The medium is collected after 24 h.
      
   4. Cells are trypsinized and alive cells are counted with Trypan blue 1:1.
      
   5. The medium collected is centrifuged at 13,000 rpm for 10 min
      
   6. The supernatant (clean medium) is diluted 1:10 in DMEM.
      
   7. 20 μl of each sample and of the standard curve (0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 mM of Lactate in DMEM) is put in wells of a 96 wells plate.
      
   8. 80 μl of assay buffer from EnzyChrom L-Lactate Assay Kit is added in each well.
      
   9. Mix well (Pipetting up and down).
      
   10. D0 is absorbed 565 nm at the beginning of the incubation ( t0) and after 20 min (t20).
       
   11. The difference of absorbtion (delta OD) is determined for the samples and the standard curve.
       
   12. The concentration of lactate for each sample is obtained from the standard curve.
       
   13.  The quantity of lactate is normalized by the final number of cells.
       

Recipes

1. Lysis buffer
   150 mM NaCl
   20 mM Na₄HPO₄/NaH₂PO₄
   10% glycerol
   1% Triton X-100 (pH 7.2)
   Complete protease inhibitors
   
2. Standard Reaction Solution
   1x Reaction Buffer
   0.1 mM DTT (dithiothreitol)
   0.5 ml 0.5 mM of D-luciferine
   2.5 μg/ml firefly luciferase and incubated at room temperature for 15 min (in the dark)
   

Acknowledgments

We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H.,  Mari, S., Qian, F., Pei, Y. and Musco, G. and the other members of the lab Boletta.

References

1. Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.