Intended Use:Assessment of cell viability is a critical step during the evaluation ofnovel drug treatments and therapies for potential cytotoxic properties
With cell viability assessment playing a central role in countless researchand environmental safety studies, there is an ever present needfor simple, straightforward analysis methods capable of distinguishingbetween live and dead cells. The Basic Calcein AM Cell Viability BioAssay™ kit allows for easy and simultaneous differentiation of live and dead cells within a single sample.
Method of Analysis:Flow cytometer, fluorescence microscope, fluorescence plate reader
Test Principle:To use Calcein AM, simply add the reagent directly to the cell sample,incubate, and analyze (no wash steps necessary). Calcein AM is a membrane permeant, fluorogenic reagent widely recognized for its utility inassessing the relative cell viability status of different cell populations. Calcein AM’s overall hydrophobic nature allows it to readily traverse the lipid bilayer structure of the cell membrane in a concentration gradient-dependent manner. Once inside the cell, the hydrophobic and non-fluorescent Calcein AM is quickly hydrolyzed by intracellularesterases that are active in live cells. This leads to the cleavage andremoval of two non-polar acetoxymethyl ester (AM) groups. Once the AM groups have been cleaved, the resulting polar (hydrophilic) and now fluorescence-capable Calcein dye molecule is efficiently retainedwithin the confines of the cell membrane. Polar dye molecules will naturally be excluded from passive diffusion back out of the cell again due to the hydrophobic lipid bilayer composition of the cell membrane. Dead cells lack active esterases and do not cleave Calcein AM. The large quantum yield of Calcein dyes enables them to be readily detected within widely used applications such as flow cytometers, fluorescence plate readers, and fluorescence microscopes. The degree of fluorescence correlates with relative cell viability status. For microscopy usage, Hoechst 33342 is included with the kit to concurrently label nuclei after labeling with Calcein. Calcein optimally excites at 494nm with maximal emission at 517nm. Hoechst 33342 can be seen using a UV-filter with excitation at 365nm and emission at 480nm.
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
