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| Product Name | Cellular ATP/ADP Ratio Bioluminescent Assay Kit, Luciferase Coupled |
| Catalog No. | CMTR-HMM-0080 |
| Description | Bioluminescent assay kit for simultaneous quantification of intracellular ATP and ADP levels and calculation of the ATP/ADP ratio — a sensitive indicator of cellular energy status, mitochondrial function, and metabolic health. The assay uses firefly luciferase, which catalyzes the ATP-dependent oxidation of D-luciferin to oxyluciferin with emission of light (peak 560 nm). In the first step, ATP is selectively measured using a stabilized luciferase/luciferin reagent. In the second step, ADP in the same well is enzymatically converted to ATP by pyruvate kinase and phosphoenolpyruvate, and the resulting ATP is measured by the same luciferase reaction. The ATP/ADP ratio in healthy cells typically ranges from 5-15 and drops below 2 during metabolic stress, apoptosis, or mitochondrial dysfunction. |
| Intended Use | Quantitative cellular energetics assessment through ATP and ADP measurement for mitochondrial toxicity screening, metabolic disease research, ischemia-reperfusion studies, drug mechanism-of-action characterization, and cell health profiling. |
| Principle / Technology | ATP-dependent firefly luciferase bioluminescence (560 nm peak); sequential measurement — ATP first, then total ATP+ADP after enzymatic ADP→ATP conversion; ADP calculated by subtraction; extremely sensitive enzyme-coupled detection. |
| Detection Method | Luminometer or luminescence microplate reader (integration time 0.5-1 second per well). |
| Sample Type | Cultured adherent and suspension cells (1×10^3-1×10^5 cells per well optimal); tissue extracts; isolated mitochondria. |
| Performance Range / Specifications | ATP linear range: 0.01-10 µM; ADP linear range: 0.02-10 µM; ATP/ADP ratio measurable from 0.1 to >100; luminescence stable for >5 minutes. |
| Sensitivity / LOD | Detection limit: 0.5 nM ATP (0.05 pmol in 100 µL); ATP/ADP ratio distinguishable with <10% CV at 1 nM each. |
| Specificity | Firefly luciferase is highly specific for ATP; pyruvate kinase specifically converts ADP to ATP; <0.01% cross-reactivity of luciferase with ADP, AMP, CTP, GTP, UTP, or dATP. |
| Reaction Conditions / Protocol | Lyse cells with ATP/ADP lysis buffer; transfer lysate to assay plate; add ATP detection reagent; read luminescence (RLU_ATP); add ADP conversion reagent; incubate 5 min; read luminescence (RLU_total); calculate [ATP] and [ADP] from standard curves. |
| Components / Formulation | Firefly luciferase/D-luciferin reagent (lyophilized), pyruvate kinase/phosphoenolpyruvate (PEP) reagent (lyophilized), ATP standard (10 µmol), ADP standard (10 µmol), cell lysis buffer, assay buffer, 96-well white luminescence plate. |
| Storage Conditions | Store luciferase reagent and standards at -20 °C (or -80 °C for extended storage); lysis buffer at 2-8 °C. |
| Shelf Life | 6 months from date of manufacture at -20 °C. |
| Package Specifications | 100 tests (ATP + ADP in duplicate), 500 tests, 1000 tests. |
| Product Form | Lyophilized enzymes and substrates; liquid standards and buffers. |
| Quality Control | Each lot tested for ATP and ADP standard curves (R² >0.99); luciferase activity >10^8 RLU per nmol ATP; ATP/ADP ratio in freshly harvested HeLa cells (expected 8-12); pyruvate kinase ADP conversion efficiency >95%. |
| Key Features | Sequential ATP then ADP measurement from same well; bioluminescent detection with ultra-high sensitivity; ATP/ADP ratio calculation; stable glow-type luminescence; compatible with high-throughput screening; includes cell lysis buffer. |
| Purity | Firefly luciferase recombinant ≥95%; D-luciferin ≥99%; ATP and ADP standards ≥99% by HPLC. |
| Concentration | ATP and ADP standards: 10 mM stocks; serially dilute in lysis buffer. |
| Activity / Unit Definition | Luciferase specific activity ≥10^9 RLU/mg protein with 1 nM ATP; pyruvate kinase ≥200 U/mg. |
| Molecular Weight | ATP: 507.18 g/mol; ADP: 427.20 g/mol. |
| Source / Origin | Recombinant Photinus pyralis (firefly) luciferase expressed in E. coli; synthetic D-luciferin; analytical grade standards. |
| pH Range / Optimal pH | Lysis buffer pH 7.4-7.8; luciferase optimal pH 7.8. |
| Shipping Conditions | Cold pack (luciferase reagent on dry ice recommended). |
| Expiration Date / Stability | 6 months at -20 °C; reconstituted luciferase reagent stable 1 week at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with adherent and suspension cells from mammalian species. White luminescence plates required for maximum sensitivity. Lysis buffer contains detergent — compatible with subsequent protein assay (BCA compatible). Avoid ATP contamination from gloves, pipette tips, or laboratory surfaces. |
| Recommended Buffer System | Tris-acetate buffer with Mg2+ for luciferase reaction, pH 7.8; cell lysis buffer contains trichloroacetic acid or detergent for rapid ATP extraction and enzyme inactivation. |
| Application Notes / Precautions | Work quickly after cell lysis — ATP is rapidly degraded by cellular ATPases. Include ATP and ADP standard curves on each plate. Normalize ATP/ADP to cell number (count parallel well) or protein content. Avoid ATP contamination from skin — wear gloves and change frequently. For adherent cells, lyse directly in culture plate. Include mitochondrial uncoupler (FCCP 10 µM, 15 min) as positive control for ATP depletion. Store ATP standard aliquots at -80 °C to prevent hydrolysis. |
| Batch-to-Batch Consistency | Luciferase light output within ±20% of reference lot per nmol ATP; pyruvate kinase ADP→ATP conversion efficiency >95% per lot. |
For research use only, not for clinical use.
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