Table 1.
Tools and Techniques to study ATP metabolism
| Tool/Technique | What it measures | How it works | References |
|---|---|---|---|
| Biochemical bulk measurements | |||
| Seahorse Extracellular Flux Analyzer (Agilent) | Glycolysis | Extracellular acidification rate (ECAR) measures change in extracellular pH | (67) |
| Seahorse Extracellular Flux Analyzer (Agilent) | Mitochondrial respiration rate | Oxygen consumption rate (OCR) measures change in oxygen concentration | (40) |
| ATP bioluminescence assay | ATP | Luciferase chemiluminescence proportional to ATP concentration | (40) |
| 13C metabolic flux analysis | Metabolic flux | stable 13C-glucose or 13C-glutamine incorporation analysis by mass spectrometry or nuclear magnetic resonance | (68) |
| Inhibitors | |||
| 2-deoxyglucose | Glycolysis | Hexokinase inhibitor | (67) |
| 3-bromopyruvate | Glycolysis | Hexokinase II inhibitor | (69) |
| Rotenone | OXPHOS | Mitochondrial complex I inhibitor | (40) |
| Thenoyltrifluoroacetone (TTFA) | OXPHOS | Mitochondrial complex II inhibitor and OXPHOS uncoupler | (70) |
| 3-nitropropionic acid | OXPHOS | Mitochondrial complex II inhibitor | (71) |
| Malonate | OXPHOS | Mitochondrial complex II inhibitor (reversible) | (72) |
| Antimycin A | OXPHOS | Mitochondrial complex III inhibitor | (72) |
| Sodium azide | OXPHOS | Mitochondrial complex IV inhibitor | (72) |
| Oligomycin | OXPHOS | Mitochondrial complex V inhibitor | (49) |
| Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazon (FCCP) | ATP synthesis | Uncoupler of mitochondrial OXPHOS by disrupting proton gradient | (40) |
| Phloretin | Glucose import | GLUT inhibitor | (67) |
| Phlorizin | Glucose import | SGLT inhibitor | (67) |
| Fluorescent dyes (excitation/emission wavelength) | |||
| Mitotracker | Mitochondrial morphology | Cell permeable, mitochondrion specific dyes. Various spectra available | (73) |
| MitoView | Mitochondrial morphology | Cell permeable, mitochondrion specific dyes. Various spectra available | (74) |
| MitoSOX | Mitochondrial superoxides | Red mitochondrial superoxide indicator (510/580) | (75) |
| TMRE/TMRM | Mitochondrial membrane potential | Red fluorescence based on mitochondrial membrane potential (549/574nm) | (73) |
| MitoProbe DiIC1(5) | Mitochondria Membrane Potential | Far red fluorescence signal increases with increased mitochondrial activity (638/658) | (76) |
| 2-NBDG/6-NBDG | Glucose | Green fluorescent glucose analog (465/540nm) | (56) |
| Genetically encoded biosensors (excitation/emission wavelength) | |||
| PyronicSF | Pyruvate | Intensiometric (488/525) | (77) |
| Laconic | Lactate | FRET ratiometric (430/485,528) | (78) |
| OGsor | 2-oxaloglutarate | FRET ratiometric (440/478,528) | (78) |
| PercevalHR | ATP:ADP | Ratiometric (405,490/530nm) | (79) |
| ATeams | ATP | FRET ratiometric (435/475,527) | (80) |
| BTeam | ATP | BRET ratiometric (no excitation required/460,527nm) | (81) |
| iATPsnFR | ATP | Intensiometric (490/512) | (66) |
| NAD+ biosensor | NAD+ | Intensiometric (488/520) | (82) |
| Peredox | NADH-NAD+ | Intensiometric (488/520) | (83) |
| Green Glifon 50, 600, 4000 | Glucose | Intensiometric (488/525) | (84) |
| FGBP | Glucose | Intensiometric (485,400/528) | (85) |
| iGlucoSnFR | Glucose | Intensiometric (488/495-575) | (86) |