ATP 和乳酸盐含量的定量测定

Isaline  Rowe; Marco  Chiaravalli; Alessandra  Boletta

doi:10.21769/BioProtoc.986

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Peer-reviewed

ATP and Lactate Quantification

ATP 和乳酸盐含量的定量测定

Isaline Rowe

Marco Chiaravalli

AB Alessandra Boletta email

发布: 2013年12月05日第3卷第23期 DOI: 10.21769/BioProtoc.986 浏览次数: 16542

评审: Anonymous reviewer(s)

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Cover of Nature Medicine, featuring study using the protocol.

Apr 2013

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Abstract

Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, pyruvate is imported into mitochondria where it is used in the Krebs (or TCA) cycle and oxydative phosphorylation. The global process of oxydative phosphorylation yields to 32 ATP per glucose molecule. For reasons not fully understood, in some pathological cases like cancer, cells use anaerobic glycolysis even in the presence of oxygen, in which case the process is called aerobic glycolysis (or Warburg effect). This results in an increased uptake of glucose and lactate production. Measure of intracellular ATP content and lactate concentrations can provide a readout of aerobic glycolyis.

Materials and Reagents

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)
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

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)
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