Advanced Calcein AM Cell Viability Kit

1. Prepare samples and controls in 0.5 mL 1X Assay Buffer or buffer of choice at a cell density between 3-5 x 105 cells/mL. If samples were cultured in serum-containing medium, wash the samples prior to adding the DAF-2DA dye., 2. Prepare DAF-2DA staining solution by adding 56 µL DAF-2DA to 444 µL water. This is sufficient volume to stain 50 x 0.5 mL or 100 x 0.25 mL samples., 3. Pre-load samples with DAF-2DA staining solution by adding 10 µL into 490 µL cultured cells, or 5 µL into 245 µL cultured cells., 4. Incubate 1 hour at 37°C., 5. Wash samples at least once with 1X Assay Buffer to remove excess dye, and then resuspend in 1X Assay Buffer., 6. Treat cells with test compounds for desired period of time to induce NOS production. Keep cells protected from light., 7. Analyze with a flow cytometer, fluorescence plate reader, or fluorescence microscope. DAF-2DA excites at 488 nm and emits at 515 nm.

Calcein AM Reagent, 1 vial, #6696, 7-Aminoactinomycin D (7-AAD) vital dye, 0.26 mg, 2 vials, #6163, Hoechst 33342, 1 vial, #639, 10X Cellular Assay Buffer, 60 mL, 1 bottle, #6695, Kit Manual

The Advanced Calcein AM Cell Viability kit developed by ImmunoChemistry Technologies, LLC (ICT) combines Calcein AM with 7-aminoactinomycin D (7-AAD) to allow for easy and simultaneous labeling of live, membrane compromised, and dead cells within a single sample. Assessment of cell viability is a critical step during the evaluation of novel drug treatments and therapies for potential cytotoxic properties. With cell viability assessment playing a central role in countless research and environmental safety studies, there is an ever present need for simple, straightforward analysis methods capable of distinguishing between live and dead cells. Calcein AM is a membrane permeant, fluorogenic, reagent widely recognized for its utility in assessing 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 intracellular esterases that are active in live cells. This leads to the cleavage and removal 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 retained within 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 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. Because Calcein alone will detect cells that are alive, but some of which could possess compromised cell membrane structure and thus be in the process of dying, it is possible to obtain an overly positive picture of the overall health status of the cell population. Loss of cell membrane integrity, often indicative of necrosis or late stage apoptosis, can be detected using the vital staining dye, 7-AAD, a red fluorescing live/dead stain. This dye easily penetrates cell membrane-compromised cells, binding tightly to GC rich regions of DNA. Combining these two different types of fluorescent cell-status-indicator reagents within a single test allows for better resolution of the live and dead cell populations by making it possible to identify the percentage of cells that are 7-AAD positive versus 7-AAD negative within the green fluorescing Calcein positive cell populations. Calcein optimally excites at 494 nm with maximal emission at 517 nm. Hoechst 33342 can be seen using a UV-filter with excitation at 365 nm and emission at 480 nm. 7-AAD can be efficiently excited at 485-495 nm, but exhibits an optimal emission Well into the red fluorescence range (647 nm). This significant difference in fluorescence emission wavelength between the green fluorescing Calcein and the red fluorescing 7-AAD live/dead dye simplifies flow cytometer gating and compensation. Live cells retaining hydrolyzed Calcein are monitored on the FL-1 channel, while membrane compromised or dead cells that have taken up 7-AAD are monitored using the FL-3 channel. Combining the Calcein AM cell viability dye with a membrane integrity dye like 7-AAD makes it easy to distinguish between live, live but membrane-compromised, and dead cells within a single sample.