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| Product Name | Calcein AM (Ultrapure Grade) |
| Catalog No. | RESK-0531 |
| Description | Calcein AM is a fluorescent staining reagent for labeling living cells, emitting green fluorescence (Ex = 490 nm, Em = 515 nm). The introduction of acetoxymethyl ester (AM) groups onto the conventional calcein scaffold increases its hydrophobicity, allowing it to readily cross the membrane of living cells. Once inside the cell, Calcein AM (itself non-fluorescent) is cleaved by intracellular esterases to form calcein, a membrane-impermeant polar molecule that is retained within the cell and emits strong green fluorescence. Compared with similar reagents such as BCECF-AM and CFDA, Calcein AM has extremely low cytotoxicity and is the fluorescent probe best suited for live cell staining; it does not inhibit cellular functions such as proliferation or lymphocyte chemotaxis. Because dead cells lack esterase activity, Calcein AM is used only for viability testing and short-term labeling of living cells. Propidium iodide (PI), a nuclear staining dye, cannot cross the membrane of living cells but penetrates the disordered membrane of dead cells to reach the nucleus and intercalates into the DNA double helix, producing red fluorescence (Ex = 535 nm, Em = 617 nm); PI therefore stains only dead cells. Since both calcein and PI-DNA can be excited at 490 nm, living and dead cells can be observed simultaneously by fluorescence microscopy, while excitation at 545 nm reveals only dead cells. Calcein AM and PI are therefore frequently combined for dual staining of live and dead cells. As optimal staining conditions vary between cell lines, the appropriate concentrations of Calcein AM and PI should be determined individually. This product is Calcein AM supplied as a powder at ultrapure grade (>=95%), and it can be used in combination with PI for simultaneous detection of living and dead cells. |
| Intended Use | Fluorescent staining of living cells for cell viability testing and short-term live cell labeling; dual staining of live and dead cells in combination with propidium iodide (PI). |
| Principle / Technology | Acetoxymethyl ester (AM) derivatization renders calcein membrane-permeant and non-fluorescent. Inside living cells, intracellular esterases hydrolyze Calcein AM to calcein, a polar, membrane-impermeant product that is retained in the cell and emits strong green fluorescence. Dead cells lack esterase activity and are not labeled, so fluorescence indicates esterase-active, membrane-intact living cells. |
| Detection Method | Fluorescence microscopy or fluorescence-based detection with Ex = 490 nm and Em = 515 nm (green fluorescence). In dual staining with PI (Ex = 535 nm, Em = 617 nm), excitation at 490 nm allows simultaneous observation of live and dead cells, while excitation at 545 nm reveals only dead cells. |
| Sample Type | Living cells from animal or plant origin (no species specificity). |
| Specificity | Labels living cells only; dead cells lacking esterase activity are not stained. As a lipophilic probe that inserts into lipid membranes, it has no specificity for particular animal or plant cell types. |
| Reaction Conditions / Protocol | Optimal staining conditions vary between cell lines; the appropriate concentrations of Calcein AM (and PI, if used in combination) should be determined individually. If the fluorescent signal appears weak, staining time and concentration can be increased. |
| Components / Formulation | Single-component fluorescent probe supplied as a powder. |
| Storage Conditions | Store dry at -20 °C; avoid direct exposure to strong light. |
| Shelf Life | 1 year under recommended storage conditions. |
| Package Specifications | 1 x 50 μg 2 x 50 μg 20 x 50 μg 1 mg |
| Product Form | Powder. |
| Purity | >=95% (HPLC), ultrapure grade |
| Molecular Weight | 994.86 |
| Source / Origin | Chemical synthesis. CAS No. 148504-34-1; molecular formula C46H46N2O23; English synonym: 3',6'-Di(O-acetyl)-4',5'-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, tetraacetoxymethyl ester. |
| Shipping Conditions | Shipped on wet ice (ice packs). |
| Expiration Date / Stability | Stable for 1 year when stored dry at -20 °C, protected from direct exposure to strong light. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Can be combined with propidium iodide (PI) for dual staining of live and dead cells; both calcein and PI-DNA are excitable at 490 nm. |
| Application Notes / Precautions | Fluorescent probes have some toxicity to living cells; complete detection and observation as soon as possible after staining. To minimize error, keep the interval between probe loading and instrument measurement as short as possible (stimulation periods excepted). If fluorescence appears weak after staining, try increasing the staining time and concentration. For observing morphological changes of living cells over several hours, lipophilic cyanine dyes such as DiI, DiO, DiD, or DiR are generally used instead. Avoid direct exposure of the product to strong light. |
For research use only, not for clinical use.
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