Cytotoxicity 1 Multiparameter Kit

• Simultaneously quantify four hallmark indicators of cytotoxicity: nuclear size/ morphology, cell membrane permeability, lysosomal mass-pH, and cell density (number of cells per field) - shortening the assay from days to hours
• Lysosomal marker makes it ideal for measuring phospholipidosis
• Multiplexed fluorescence-based assay provides sensitive quantitative and qualitative results without radioactivity
• No cell lysis, purification, or filtration steps—saving time and labor

The Multiparameter Cytotoxicity 1 Kit includes reagents for identifying changes in nuclear morphology and size, membrane permeability status, lysosome mass/pH changes and cell density changes (number of cells per field) caused by compound toxicity. When used with the Multiparameter Cytotoxicity 1 BioApplication software on the Thermo Scientific ArrayScan® HCS Reader, the kit can be used as a cell health assay where, for nuclear morphology, cell permeability and lysosomal physiology parameters, the number and percentage of cells in the population that are beyond the normal physiological range can be quantified. Additionally, the assay can be used to identify compounds affecting cell population density.

Depending on the type of toxic insult, cells often undergo either necrosis or apoptosis, accompanied by changes in nuclear size or morphology. The Hoechst 33342 dye used in this assay labels DNA and emits a blue fluorescence. This nuclear stain identifies individual nuclei and measures changes in nuclear size and morphology that result from toxic insult.

The cell membrane maintains cellular homeostasis, providing a specialized environment different from its extracellular surroundings, and providing a mechanism for the controlled exchange of its nutrients with its surroundings. Certain toxins can affect cell membrane integrity leading to the cell becoming permeable, eventually causing cell death. Healthy cells are nearly impermeable to the indicator dye used in this assay; however, after compromising the cell membrane’s permeability, the dye stains the nucleus with a green fluorescence.

Physiologically healthy cells have intracellular organelles with characteristic properties, including lysosomes and endosomes that maintain specific internal acidic pH ranges for cells to function normally. Toxins can interfere with cell functionality by affecting the pH of these organelles or by causing a change in lysosom number. The dye used in the assay to determine changes to lysosomal physiology is a weak base that accumulates in acidic organelles such as lysosomes and endosomes. A decrease in pH of acidic organelles or increase in lysosome numbers by compound toxicity results in an increase of fluorescence intensity. Conversely, an increase in pH of these organelles or decrease in lysosome numbers of results in a decrease in staining intensity of the dye.

After labeling, cells are fixed with formaldehyde and scanned and analyzed with the ArrayScan HCS Reader and the Multiparameter Cytotoxicity 1 BioApplication software. The assay was developed, optimized and validated in the human hepatoma cell line HepG2. In addition to the liver and kidney, other target organs/tissues for xenobiotics (toxicity effects) could be the lungs, central nervous system, heart, immune system and even the skin. With minor protocol changes, this assay can be easily adapted for use with any cell types/lines that originate from the organs/tissues listed.

Figure 1. HepG2 cells treated with 100 µM valinomycin for 24 hours (right), and untreated (left). Nuclei appear in blue, lysosomes, in red, cell membrane impermeant dye in green.  All cells in right panel are dead, as indicated by co-localized nuclear and cell impermeant dye. Images taken from the ArrayScan HCS Reader.

Figure 2. Dose response curves for the three parameters of cytotoxicity measured simultaneously in HepG2 cells treated with valinomycin for 24 hr.

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