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| Product Name | CellTiter-Fluor Cell Viability Assay (Protease Activity-Based) |
| Catalog No. | CATR-HMM-0057 |
| Description | A homogeneous, single-addition fluorometric cell viability assay based on the measurement of a conserved, constitutive protease activity within viable cells. A cell-permeable fluorogenic peptide substrate (GF-AFC) enters intact cells and is cleaved by live-cell protease activity to generate a fluorescent signal proportional to the number of viable cells. The signal is stable for several hours, enabling batch plate processing in high-throughput screening formats. |
| Intended Use | Multiplexed cell viability measurement preceding downstream assays (luminescence, reporter gene, or extraction-based assays); cytotoxicity screening; cell proliferation quantification; normalization of gene reporter assays; high-throughput viability assessment. |
| Principle / Technology | Live-cell permeant fluorogenic peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin, GF-AFC) enters viable cells and is cleaved by constitutive aminopeptidase activity within live cells, releasing fluorescent AFC (7-amino-4-trifluoromethylcoumarin); signal is proportional to viable cell number |
| Detection Method | Fluorometric; excitation 380–400 nm, emission 490–510 nm |
| Sample Type | Adherent and suspension mammalian cells; compatible with most cell lines including HEK293, HeLa, CHO, Jurkat, and primary cells |
| Sensitivity / LOD | Detects as few as 50 cells per well (96-well format); linear range 50–50,000 cells per well; Z' factor >0.7 for screening |
| Specificity | Specific for viable cell aminopeptidase activity; signal absent in dead cells and cell-free medium; no cross-reactivity with common media components |
| Reaction Conditions / Protocol | Add reagent at equal volume to culture medium; mix by orbital shaking 30 seconds; incubate 30–60 minutes at 37 °C; read fluorescence; no washing or medium removal required |
| Components / Formulation | GF-AFC substrate (100× concentrate in DMSO), assay buffer; ready-to-use after dilution |
| Storage Conditions | Store at -20 °C; protect from light; substrate is light-sensitive |
| Shelf Life | 6 months at -20 °C from manufacture date |
| Package Specifications | 10 mL (100 assays), 50 mL (500 assays), 100 mL (1,000 assays) |
| Product Form | Concentrated fluorogenic substrate in DMSO; diluted in assay buffer before use |
| Key Features | Non-lytic — cells remain viable for downstream multiplex assays; simple add-mix-read protocol; signal stable 2–6 hours; compatible with subsequent luminescence assays (multiplex viability + reporter gene); wider linear range than resazurin-based assays; minimal interference from test compounds compared to reductase-based assays |
| Purity | GF-AFC purity ≥98% by HPLC; endotoxin <0.05 EU/mL in working solution |
| Concentration | As specified on product label; working concentrations optimized per protocol |
| Activity / Unit Definition | Signal-to-noise ratio and linearity verified on reference cell lines per lot |
| Molecular Weight | Varies by dye or reagent component as specified |
| Source / Origin | Synthetic dyes and tetrazolium compounds; recombinant enzymes where applicable |
| pH Range / Optimal pH | pH 7.2–7.4 for cell-based assays |
| Shipping Conditions | Cold pack -20 °C; protect from light; avoid freeze-thaw |
| Expiration Date / Stability | 6 months at -20 °C; reconstituted working reagent stable 24 hours at 4 °C protected from light; do not refreeze reconstituted reagent |
| Regulatory / Compliance | For laboratory and research use only; RUO; manufactured under ISO 9001; not classified as dangerous goods |
| Compatibility | Compatible with phenol red-containing and serum-containing media; phenol red may increase background fluorescence — use phenol red-free medium and include cell-free control wells; DMSO concentrations up to 0.5% do not interfere |
| Recommended Buffer System | PBS or phenol red-free complete medium as specified in protocol |
| Application Notes / Precautions | For multiplex assays, measure CellTiter-Fluor first (non-lytic), then proceed with secondary assay (e.g., Caspase-Glo for apoptosis, or reporter gene luciferase). Include cell-free blanks and a cell dilution series for standard curve. For suspension cells, allow cells to settle 10 minutes after reagent addition before reading. Pre-warm reagent to 37 °C for 15 minutes before addition to avoid thermal shock. |
| Batch-to-Batch Consistency | Signal linearity R² ≥0.99 with reference cell number standard curve per lot |
For research use only, not for clinical use.
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