Figure 1. HeLa cells were seeded at 1,500 to 100,000 cells/well in a 96-well black plate with clear bottom and incubated overnight. Cells were exposed to Triton™ X-100 (0.05%, 10 min) and cell viability/toxicity was measured using the Cell Health Assay Kit #13837. Relative fluorescence for Calcein-AM (left) and Propidium Iodide (right) are shown.
Figure 2. Treatment of HeLa cells (4 x104 cells/well) with increasing concentrations of terfenadine (4 hr) results in reduced cell viability as detected by the Cell Health Assay Kit.
Figure 3. Flow cytometric analysis of Jurkat cells, untreated (green) or terfenadine-treated (50 μM, 4 hr; red), using Calcein-AM (0.1 μM, 20 min) and Propidium Iodide (3 μM, 20 min).
Figure 4. Fluorescent analysis of HeLa cells (4x104 cells/well) treated with terfenadine (12.5 μM, 4 hr) and stained with Calcein-AM (1 μM, 30 min; green) and Propidium Iodide (3 μM, 30 min; red).
The Cell Health Assay Kit is a fluorescence-based assay that determines cell viability by measuring intracellular esterase activity and plasma membrane integrity. The kit contains Calcein-AM, which stains viable cells, and Propidium Iodide (PI), an indicator of dead cells. Calcein-AM is the acetomethoxy form of calcein, a highly lipophilic, cell membrane permeable dye. Intracellular esterase converts the non-fluorescent Calcein-AM to the highly fluorescent calcein, which is retained within live cells and produces an intense green fluorescence. The DNA-binding agent Propidium Iodide is cell membrane impermeable and only enters dead cells or those with damaged cell membranes. Intracellular PI binds DNA and undergoes an approximate 40-fold enhancement in fluorescence intensity. As a result, live cells will produce a strong green fluorescence resulting from the conversion of Calcein-AM to calcein, while dead cells produce a strong red fluorescence due to the presence of Propidium Iodide.
At the recommended reagent concentrations and volumes, one kit will provide for 1000 assays (96-well plate format) or 200 flow cytometry assays.
The Cell Health Assay Kit will detect cell viability/toxicity in most eukaryotic cells. Between 500 and 50,000 cells/well can be used in this assay. A cell number titration is recommended when using a plate-reader with 96-well plate. For optimal conditions, titrations of Calcein-AM and Propidium Iodide are recommended for each cell line.
Measures of cell viability and cytotoxicity are broadly used to study the effects of growth factors and cytokines, inhibitors and activators, and immune response signals. Many viability and toxicity assay kits (such as XTT, resazurin, and BrdU cell kits) monitor the presence or viability of live cells by measuring the metabolic conversion or incorporation of a substrate into cellular molecules. Assay kits that measure dead or dying cells usually rely on the activity of apoptotic enzymes (i.e. caspase-3) or the intracellular release of an enzyme or molecule indicative of cell death (i.e. cytochrome c, LDH). The Cell Health Assay Kit is able to stain both live and dead cells simultaneously by using two fluorescent probes. After treating cells with a combination of Calcein-AM and Propidium Iodide, live cells will produce strong green fluorescence while dead cells produce strong red fluorescence (1,2). These fluorescent signals can be detected using multiple methods, including a plate reader or scanner, flow cytometer, or fluorescent microscope. This dual assay is specifically designed to assess viability and toxicity for eukaryotic cells (1,2), but may also be used to assay some bacteria or yeasts (3,4).
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