Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. The determination of cell viability depends on these physical and biochemical properties of cells. Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method. Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non-fluorescent before interacting with cells.
• Calcein AM (Component A)
• Ethidium homodimer-1 (Component B)
Storage and Handling of Reagents
Reagents in this kit should be stored sealed, desiccated, protected from light and frozen at ≤–20˚C. Allow the reagents to warm to room temperature and centrifuge briefly before opening. Before refreezing, seal all stock solutions tightly. Calcein AM is susceptible to hydrolysis when exposed to moisture. Prepare aqueous working solutions containing calcein AM immediately prior to use, and use within one day. EthD-1 is stable and insensitive to moisture. Stock solutions of EthD-1 in DMSO/ H2O or other aqueous media can be stored frozen at ≤–20°C for at least one year.
500tests ; 1000tests
Celltechgen provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please request the Safety Data Sheet (SDS) provided for the product.