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| Product Name | Calcein-AM Cell Viability Assay Kit, Green Fluorescence, Live Cell Staining |
| Catalog No. | CATR-HMM-0059 |
| Description | Fluorometric cell viability assay kit based on Calcein-AM (calcein acetoxymethyl ester), a non-fluorescent hydrophobic compound that passively crosses live cell membranes. Intracellular esterases in viable cells cleave the acetoxymethyl ester groups, converting Calcein-AM to green-fluorescent calcein (Ex/Em 495/515 nm) that is retained within cells due to its polyanionic nature. Dead cells lacking active esterases do not produce fluorescent signal. The intensity of green fluorescence is directly proportional to the number of viable cells, enabling quantitative viability assessment by fluorescence microscopy, flow cytometry, or microplate reader. |
| Intended Use | Quantitative measurement of live cell number in cell viability, proliferation, cytotoxicity, and chemosensitivity assays for drug discovery, toxicology screening, and basic cell biology research. |
| Principle / Technology | Acetoxymethyl ester derivatization enables passive membrane permeation; intracellular esterase hydrolysis releases fluorescent calcein trapped within viable cells; non-fluorescent substrate minimizes background. |
| Detection Method | Fluorescence microplate reader (Ex/Em 485/530 nm), fluorescence microscopy (FITC filter set), or flow cytometry (488 nm excitation, 530/30 nm emission). |
| Sample Type | Adherent and suspension cultured mammalian cells; primary cells from various tissues and species. |
| Performance Range / Specifications | Linear detection range: 100-100,000 viable cells per well (96-well plate); dynamic range >3 orders of magnitude; correlation with trypan blue exclusion R² >0.98. |
| Sensitivity / LOD | Detection limit approximately 50 viable cells per well in 96-well format (200 µL volume); signal-to-background ratio >100:1 at 10,000 cells/well. |
| Specificity | Calcein-AM selectively reports viable cells with active esterase activity; does not stain dead cells, bacteria, or yeast at recommended concentrations; minimal background from serum-containing media. |
| Reaction Conditions / Protocol | Add Calcein-AM working solution (2 µM) to cells; incubate 30-45 minutes at 37 °C; measure fluorescence without washing (optional wash to remove extracellular dye). |
| Components / Formulation | Calcein-AM (1 mg, lyophilized), anhydrous DMSO (1 mL), 10× Assay Buffer (25 mL), detailed protocol. |
| Storage Conditions | Store Calcein-AM at -20 °C desiccated and protected from light; assay buffer at 2-8 °C. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 mg (sufficient for ~500-1000 tests in 96-well format), 5 mg bulk size. |
| Product Form | Lyophilized pale yellow powder; colorless liquid after reconstitution. |
| Quality Control | Each lot tested for esterase substrate activity using purified porcine liver esterase; fluorescence intensity verified in live/dead HeLa cell mixtures (0-100% viability series). |
| Key Features | Single-step homogeneous assay (no wash required); low cytotoxicity allows kinetic monitoring; compatible with serum-containing media; brighter and less pH-sensitive than BCECF-AM and CFDA-SE; suitable for high-throughput screening. |
| Purity | Calcein-AM purity ≥95% by HPLC; free calcein <1%. |
| Concentration | Prepare 1 mM stock in DMSO; use at 0.5-5 µM final concentration depending on cell type. |
| Activity / Unit Definition | Fluorescence quantum yield of calcein ~0.75 in aqueous buffer at pH 7.4. |
| Molecular Weight | Calcein-AM: 994.86 g/mol (C46H46N2O23). |
| Source / Origin | Synthetic fluorescein derivative manufactured under controlled laboratory conditions. |
| pH Range / Optimal pH | Optimal fluorescence at pH 7.0-8.0; calcein fluorescence is pH-dependent with pKa ~6.8. |
| Shipping Conditions | Ambient temperature; protect from moisture and light. |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted 1 mM DMSO stock stable for 1 month at -20 °C in sealed, desiccated container. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic procedures. |
| Compatibility | Compatible with DMEM, RPMI-1640, MEM, and other standard culture media with or without phenol red (phenol red does not interfere at 530 nm emission). Compatible with most drug compounds at screening concentrations; verify with vehicle control. |
| Recommended Buffer System | 10× Assay Buffer: PBS with calcium and magnesium, pH 7.4. |
| Application Notes / Precautions | Pre-warm Calcein-AM working solution to 37 °C before adding to cells. Vortex briefly after adding to ensure homogeneous staining. For adherent cells, ensure dye solution covers entire monolayer. Include cell-free wells (buffer only) as blank controls. Calcein-AM is light-sensitive — prepare and use under subdued lighting. Do not store diluted working solution; prepare fresh each time. |
| Batch-to-Batch Consistency | Calcein-AM content within ±5% of label; free calcein <1%; esterase substrate activity within ±10% of reference lot. |
For research use only, not for clinical use.
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