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| Product Name | Calcein-AM/PI Double Stain Kit (Live/Dead Cell Staining) |
| Catalog No. | RESK-0145 |
| Description | Calcein-AM is a fluorescent cell staining reagent for labeling live cells, emitting green fluorescence (Ex = 490 nm, Em = 515 nm). The introduction of an acetoxymethyl (AM) ester group onto traditional Calcein increases its hydrophobicity, allowing it to readily cross the membrane of live 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. Because of its extremely low cytotoxicity, Calcein-AM is one of the most suitable fluorescent probes for live-cell staining and does not impair any cellular functions, such as proliferation or lymphocyte chemotaxis. Since dead cells lack esterase activity, Calcein-AM is used for viability testing and short-term labeling of live cells only. It is therefore commonly combined with a dead-cell fluorescent probe such as propidium iodide (PI) for simultaneous fluorescent double staining of live and dead cells. PI cannot cross the membrane of live cells; it only passes through disordered membrane regions of dead cells to reach the nucleus, where it intercalates into the DNA double helix and produces red fluorescence (Ex = 535 nm, Em = 617 nm). Because both Calcein and PI-DNA can be excited at 490 nm, live and dead cells can be observed simultaneously by fluorescence microscopy; excitation at 545 nm reveals dead cells only. |
| Intended Use | Simultaneous fluorescent double staining and analysis of live and dead cells for cell viability and cytotoxicity assessment. For research use only. |
| Principle / Technology | Dual-dye principle: Calcein-AM permeates live cell membranes and is hydrolyzed by intracellular esterases into fluorescent, membrane-impermeant Calcein (green), labeling live cells; propidium iodide (PI) enters only membrane-compromised dead cells and intercalates into nuclear DNA (red). Cells showing both green and red fluorescence are judged as dead, since dead cells lack esterase activity and produce little or no Calcein. |
| Detection Method | Fluorescence microscopy or flow cytometry. Calcein (live cells): Ex = 490 nm, Em = 515 nm (FITC/FL1 channel). PI (dead cells): Ex = 535 nm, Em = 617 nm (FL3 channel). Both dyes can be excited at 490 nm for simultaneous observation; 545 nm excitation shows dead cells only. |
| Sample Type | Cultured cells (cell suspensions or adherent cells grown on glass dishes/chambers). Cells in confocal dishes can be stained directly without trypsinization. |
| Performance Range / Specifications | Optimized for staining 200 uL of cell suspension per test; sufficient for 500 tests (500 T) or 1000 tests (1000 T). |
| Specificity | Calcein-AM stains only live cells with intact esterase activity (strong green fluorescence); PI stains only dead cells with compromised membrane integrity (red nuclear fluorescence). Cells positive for both colors are judged as dead. |
| Reaction Conditions / Protocol | 1. Prepare cell suspension (200 uL per test) or grow cells on glass-bottom dishes. 2. Add Calcein-AM and PI working solutions to the sample (optimize staining concentrations of both dyes before formal experiments). 3. Incubate and observe directly by fluorescence microscopy, or analyze by flow cytometry (FL1 for Calcein, FL3 for PI). |
| Components / Formulation | Component A: Calcein-AM solution. Component B: Propidium iodide (PI) solution. Component C: assay buffer. |
| Storage Conditions | Components A and B: store at -20 °C, protected from light and kept dry. Component C: store at -20 °C; may be kept at 4 °C if used frequently. |
| Shelf Life | 1 year. |
| Package Specifications | 500 T 1000 T |
| Product Form | Liquid kit components. |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | 1 year under recommended storage conditions. |
| Compatibility | Compatible with fluorescence microscopy and flow cytometry. Direct observation of adherent cells in multi-well plastic plates is not recommended (plastic affects light transmission and gives poor results); use glass-bottom dishes instead. |
| Application Notes / Precautions | Optimize the staining concentrations of Calcein-AM and PI before formal experiments. Protect dye solutions from light. For adherent cells, glass dishes are recommended rather than plastic multi-well plates. |
For research use only, not for clinical use.
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