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| Product Name | Fatty Acid Oxidation Assay Kit, Fluorometric, Cell-Based |
| Catalog No. | CMTR-HMM-0082 |
| Description | Cell-based fluorometric assay kit for real-time measurement of fatty acid oxidation (FAO) rates in intact cultured cells using a palmitate-BSA conjugate and an oxygen-sensitive phosphorescent probe. The assay measures the rate of oxygen consumption specifically attributable to mitochondrial β-oxidation of long-chain fatty acids. Cells are incubated with palmitate-BSA as the primary metabolic substrate, and an oxygen-sensing phosphorescent probe embedded in the assay medium reports oxygen consumption in real time. The oxygen consumption rate (OCR) specifically attributable to FAO is quantified by comparing OCR in the presence and absence of the CPT-1 inhibitor etomoxir, which blocks the rate-limiting step of long-chain fatty acid entry into mitochondria. |
| Intended Use | Measurement of mitochondrial long-chain fatty acid β-oxidation capacity in intact cells for metabolic research, obesity and diabetes studies, cancer metabolism (fatty acid utilization), and drug effects on lipid metabolism. |
| Principle / Technology | Palmitate-BSA provides fatty acid substrate; CPT-1 transports fatty acyl-CoA into mitochondria; O2 consumption by ETC is measured by phosphorescent O2 probe (lifetime or intensity mode); etomoxir inhibitable O2 consumption represents FAO-specific respiration; real-time kinetic monitoring. |
| Detection Method | Fluorescence lifetime or intensity plate reader with O2-sensing capability (Ex/Em 380/650 nm); time-resolved fluorescence mode for optimal sensitivity. |
| Sample Type | Intact adherent cultured cells (primary hepatocytes, cardiomyocytes, myotubes, cancer cell lines); cells must be in glucose-limited medium to favor FAO. |
| Performance Range / Specifications | OCR range: 1-100 pmol O2/min/10^6 cells; FAO-specific OCR 10-80% of total OCR depending on cell type and conditions; linear response for 2-4 hours at 37 °C. |
| Sensitivity / LOD | Detection of FAO-specific OCR change as low as 0.5 pmol O2/min/10^6 cells; distinguishable etomoxir inhibition of 10% or greater in total OCR. |
| Specificity | Etomoxir specifically inhibits CPT-1, blocking long-chain fatty acid entry into mitochondria; residual OCR after etomoxir represents non-FAO respiration (glucose, glutamine, short-chain fatty acid oxidation). |
| Reaction Conditions / Protocol | Seed cells in assay plate; replace medium with FAO assay medium (low glucose, palmitate-BSA, O2 probe); add etomoxir to control wells; seal with mineral oil overlay; measure OCR kinetically at 37 °C for 1-4 hours; calculate FAO-OCR = total OCR - etomoxir-resistant OCR. |
| Components / Formulation | Palmitate-BSA conjugate (2 mM), etomoxir (10 mM in DMSO), O2-sensing phosphorescent probe, FAO assay medium (low glucose base), mineral oil (for oxygen barrier), 96-well black microplate with clear bottom. |
| Storage Conditions | Store palmitate-BSA at -20 °C; etomoxir at -20 °C; O2 probe at 2-8 °C protected from light; assay medium at 2-8 °C. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 1 × 96-well kit (includes ~50 test wells in triplicate with controls). |
| Product Form | Palmitate-BSA: sterile liquid; etomoxir: DMSO solution; O2 probe: concentrated DMSO or aqueous solution. |
| Quality Control | Each lot tested for etomoxir inhibition efficiency (>80% reduction of palmitate-driven OCR in HepG2 cells); palmitate-BSA molar ratio verified (6:1); O2 probe phosphorescence lifetime calibration. |
| Key Features | Cell-based intact cell FAO measurement; real-time oxygen consumption monitoring; etomoxir control for FAO specificity; palmitate-BSA physiological substrate; compatible with standard fluorescence plate readers with TRF capability; non-destructive. |
| Purity | Palmitate ≥99%; BSA fatty acid-free ≥98%; etomoxir ≥98% by HPLC; O2 probe purity verified. |
| Concentration | Palmitate-BSA: 2 mM stock (palmitate concentration); etomoxir: 10 mM stock; use at 2 mM palmitate and 40 µM etomoxir final. |
| Activity / Unit Definition | O2 probe phosphorescence lifetime 60-70 µs in air-saturated buffer; Stern-Volmer quenching constant ~300 M-1. |
| Molecular Weight | Palmitate: 256.42 g/mol; etomoxir: 323.82 g/mol (C15H21ClO4 · C2H8NO). |
| Source / Origin | Synthetic palmitate and etomoxir; BSA from bovine plasma; Ru(II)-based phosphorescent O2 probe. |
| pH Range / Optimal pH | Assay medium pH 7.4. |
| Shipping Conditions | Cold pack (palmitate-BSA and etomoxir on dry ice recommended). |
| Expiration Date / Stability | 6 months at recommended storage; palmitate-BSA avoid freeze-thaw; etomoxir stable for 3 months at -20 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. Etomoxir is a research compound — not for human use. |
| Compatibility | Compatible with adherent cell types. Cells must be capable of fatty acid oxidation — hepatocytes, cardiomyocytes, and skeletal myocytes have high FAO capacity. Most cancer cell lines have low FAO under standard culture conditions. Use low glucose (2.5 mM) assay medium to minimize Crabtree effect. |
| Recommended Buffer System | FAO assay medium: DMEM base with 2.5 mM glucose, 0.5 mM carnitine, 25 mM HEPES, pH 7.4. |
| Application Notes / Precautions | Optimize cell seeding density for each cell type — typically 2-5×10^4 cells/well for hepatocytes; over-confluence causes hypoxia throughout well. Pre-incubate cells in substrate-limited medium for 1-2 hours before assay to deplete endogenous substrates. The mineral oil overlay is critical — it prevents atmospheric O2 diffusion into wells. Include FCCP (1 µM) wells as positive control for maximal respiration. For cell number normalization, stain nuclei with Hoechst post-assay. |
| Batch-to-Batch Consistency | Palmitate-BSA conjugation ratio 6:1 ± 0.3; etomoxir content within ±10% of specification; O2 probe calibration verified per lot. |
For research use only, not for clinical use.
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