Cell Vitality Assay
Detection of Cell Vitality and Thiol Levels Using VitaBright-48™
- Easy to use assay evaluates cellular health and oxidative stress
- Direct detection of oxidative stress (redox state)
- Total assay takes less than one minute
- Acquisition and analysis at the single cell level in one simple step
- User-friendly protocol with predefined settings
- Clear data presentation with Plot Manager feature
- Automated PDF reports
- Export data in FCS/ACS formats
Overview of the Cell Vitality Assay
The cell vitality assay provides an easy way to determine the level of reduced thiols, such as glutathione (GSH). GSH is involved in many cellular processes including quenching free radicals, drug detoxification, cell signaling and cell proliferation.
GSH is the most abundant low molecular weight thiol in animal cells. It is present in a reduced state as GSH and an oxidized state as glutathione disulfide (GSSG i.e. two GSH molecules bound by a disulfide bridge).
In healthy cells, more than 90% of total glutathione is in the GSH state, but in disease conditions e.g. cancer, cystic fibrosis, diabetes, HIV, neurodegenerative diseases and aging, there is a depletion of the GSH pool, leading to oxidative stress of the cell. Oxidative stress is an early indicator of apoptosis, i.e. programmed cell death. During apoptosis, the levels of GSH decrease and can be detected using the VitaBright-48™ fluorophore.
VitaBright-48™ is a patented fluorophore which only fluoresces when bound to reduced thiols (GSH). VitaBright-48™ immediately reacts with thiols when introduced to the cell sample, forming a fluorescent product. By quantifying the fluorescence, it is possible to determine the level of cellular thiols, and thus determine cellular health.
Scatter plots and histograms showing thiol level distribution in the cell population, measured as VitaBright-48™ intensity, automatically display after analysis. By comparing the VitaBright-48™ intensity of treated and control cell samples, the fraction of cells with low vitality e.g. apoptotic or stressed cells, can be determined.
Results Presented in Plot Manager
Hybridoma cells were grown in the absence (upper row) or in the presence (lower row) of etoposide, which is a common chemotherapeutic agent. Cells were stained with VitaBright-48™, acridine orange (AO) and propidium iodide (PI) and analyzed using the Vitality Assay and a NucleoCounter® NC-3000™.
Scatter plots and histograms were obtained using the NucleoView™ NC-3000™ software. Polygons and markers in the plots displayed to demarcate the various cell populations. In this example, etoposide causes a massive decrease in the level of thiols in a subpopulation (i.e. cells with low VitaBright-48™ intensity), meaning its presence decreases cellular vitality to a large extent.
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