Cytotoxicity 3 Multiparameter Kit

The kit contains a Hoechst dye, cell permeability dye, mitochondrial membrane potential dye, and a mouse monoclonal antibody against cytochrome c and a goat anti-mouse DyLight™ 649-conjugated Secondary Antibody, and various other essential reagents and buffers.

Screening potential drugs for toxicity is an essential aspect of the drug discovery process. In vitro toxicity assessments performed early in drug discovery are cost-effective and fast. Cytotoxicity is a complex process affecting multiple parameters and pathways. After toxic insult, cells often undergo either apoptosis or necrosis accompanied by changes in nuclear morphology, cell permeability and mitochondrial function, resulting in loss of mitochondrial membrane potential and release of cytochrome c from mitochondria. A goal of in vitro toxicity testing is to detect the lowest dose of a compound that causes toxicity. Because of diverse action mechanisms with different compounds, monitoring multiple, independent toxicity indicators in the same cell increases the predictive power of the assay.

Cell-based high-content screening (HCS) assays enable quantitative measurements of multiple parameters related to cytotoxicity. This kit enables simultaneous measurements in the same cell of six independent parameters that monitor cell health, including cell loss, nuclear size and morphological changes, mitochondrial membrane potential changes, cytochrome c release, and changes in cell permeability. The Hoechst dye enables monitoring of cell loss, nuclear morphology changes and DNA content, which is proportional to the total Hoechst intensity per nucleus. The other three parameters are monitored by separate dyes.

Figure 1. Staining of HepG2 cells. Cell loss, changes in nuclear size and morphology, DNA content, in mitochondrial membrane potential and cell permeability changes, and cytochrome c release were measured simultaneously. Cells were plated in 96-well collagen I-coated plates and treated with either vehicle (DMSO) or 120 µM valinomycin for 24 hours. Cells were fixed and stained according to the kit protocol. Treatment with valinomycin results in cell loss, nuclear condensation, increased total nuclear intensity, increased cell permeability, loss of mitochondrial membrane potential and cytochrome c release.

 
Figure 2. Dose-response curves. HepG2 cells were treated with various doses of valinomycin. Cells were fixed and stained according to the kit protocol.

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