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| Product Name | Seahorse XF Cell Mito Stress Test Kit (XF Analysis Reagents) |
| Catalog No. | CMTR-HMM-0059 |
| Description | A complete reagent kit for performing mitochondrial function profiling on the Seahorse XF extracellular flux analyzer platform. The kit includes the mitochondrial inhibitors oligomycin, FCCP, and rotenone/antimycin A in a pre-measured format to sequentially interrogate key parameters of mitochondrial respiration including basal respiration, ATP-linked respiration, maximal respiratory capacity, and spare respiratory capacity. |
| Intended Use | Comprehensive profiling of mitochondrial bioenergetic function in live cells for metabolic disease research, drug-induced mitochondrial toxicity screening, cancer metabolism investigation, and immunometabolism studies. |
| Principle / Technology | The Seahorse XF platform uses solid-state sensor probes to measure oxygen consumption rate (OCR, an indicator of mitochondrial respiration) and extracellular acidification rate (ECAR, an indicator of glycolysis); sequential injection of modulators reveals specific mitochondrial parameters: oligomycin inhibits ATP synthase (complex V) to determine ATP-linked respiration, FCCP uncouples the proton gradient to stimulate maximal respiration, and rotenone/antimycin A combination blocks complex I and complex III to quantify non-mitochondrial oxygen consumption. |
| Detection Method | Extracellular flux analysis using Seahorse XF analyzers (XF96, XF24, or XFe96); oxygen consumption rate (OCR) measured in pmol/min; extracellular acidification rate (ECAR) measured in mpH/min; data analyzed with Seahorse Wave software |
| Sample Type | Live adherent mammalian cells (including primary cultures, iPSC-derived cells, and cancer cell lines) seeded in Seahorse XF cell culture microplates; immune cells immobilized using Cell-Tak cell adhesive; isolated mitochondria preparations |
| Performance Range / Specifications | Respiratory parameter resolution: basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial oxygen consumption; oligomycin final concentration 1–2 μM; FCCP 0.5–2 μM (requires optimization per cell type); rotenone/antimycin A 0.5 μM each |
| Sensitivity / LOD | Detects changes in OCR of approximately 5–10% relative to baseline with optimized cell density and compound concentrations; measurable in as few as 5,000 cells per well for highly oxidative cell types |
| Specificity | Each mitochondrial modulator targets specific electron transport chain complexes with well-characterized mechanisms: oligomycin is specific for the F0 subunit of ATP synthase; FCCP is a protonophore that specifically dissipates the mitochondrial inner membrane proton gradient; rotenone inhibits complex I, antimycin A inhibits complex III, providing complete blockade of mitochondrial respiration |
| Reaction Conditions / Protocol | Seed cells in Seahorse XF microplate at optimized density (typically 10,000–80,000 cells/well for XF96); incubate overnight; on assay day, replace growth medium with XF Assay Medium (unbuffered DMEM with glucose, glutamine, pyruvate, pH 7.4); incubate in non-CO2 incubator at 37°C for 45–60 minutes; load Mito Stress Test compound ports with oligomycin, FCCP, rotenone/antimycin A; run standard Mito Stress Test protocol (3 basal measurements, 3 post-drug measurements each, 3 min mix/3 min measure cycles) |
| Components / Formulation | XF Cell Mito Stress Test kit: pre-weighed vials of oligomycin, FCCP, rotenone, and antimycin A; XF Assay Medium powder or liquid supplement; XF Calibrant solution for sensor cartridge hydration; pre-configured assay template in Wave software |
| Storage Conditions | Lyophilized compounds: -20°C in desiccated pouch, stable 24 months; reconstituted compounds: single-use recommended due to oxidation sensitivity; Assay Medium supplement: 2–8°C for 12 months |
| Shelf Life | 24 months from date of manufacture for lyophilized compounds |
| Package Specifications | 6 pouches (18 vials total, 6 assays each), 18 pouches (54 vials total, 18 assays) |
| Product Form | Pre-weighed and color-coded individual compound vials for single-assay use |
| Quality Control | Each lot tested on the Seahorse XF platform using reference cell lines (C2C12 myoblasts, HepG2 cells) to confirm expected OCR responses to each modulator; compound purity verified by HPLC; prepared in proprietary formulations to ensure solubility and stability during assay |
| Key Features | Gold-standard platform for functional mitochondrial characterization; simultaneous measurement of OCR and ECAR from the same cells; pre-measured single-use vials eliminate compound preparation errors; optimized compound formulations enhance solubility and stability; Wave software provides automated data analysis and standardized result parameters |
For research use only, not for clinical use.
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