CANCER CELL ASSAYS IN VITRO: Cell viability, Cytotoxicity and Apoptosis
Cell viability assay
Cell viability is measured using the CellTiter-GloTM luminescent cell viability assay (CTG), which determines the metabolism of viable cells in culture, based on quantitation of ATP. Cell viability assays can be multiplexed with other assays including cytotoxicity and apoptosis measurements. CellTiter-GloTM assay is an excellent assay to assess the effects of small molecules on cell proliferation and to obtain consistent and reproducible results.
Cytotoxicity can be measured using the CellToxTM Green fluorescence-based cytotoxicity assay or CytoTox-GloTM cytotoxicity assay. The CellToxTM Green assay measures changes in membrane integrity that occur as a result of cell death after experimental manipulation. The assay system uses a proprietary asymmetric cyanine dye that is excluded from viable cells but preferentially stains the DNA from dead cells. The dye is non-toxic to cells, allowing cytotoxicity assessment prior to CTG assay in the same well.
CytoTox-GloTM assay is based on luminescent detection. The assay measures extracellular protease activity when the protease is released from cells after the cell membrane integrity is compromised.
Apoptosis can be measured using the Caspase-Glo 3/7 luminescent assay, which measures caspase-3 and -7 activities. These caspases play key effector roles in apoptosis. The assay provides a proluminescent caspase-3/7 substrate. Following caspase cleavage of the substrate, free aminoluciferin is released, resulting in the production of light that can be measured. Luminescence is proportional to the amount of caspase activity present.
SCRATCH WOUND MIGRATION AND INVASION ASSAYS
Scratch wound assay also known as wound healing is a technique used to study cellular migration in 2D and cell invasion in 3D. Cell migration is linked to many biological and pathological processes such as tissue re-organization/wound repair, immune cell trafficking, chronic inflammation and tumor metastasis which makes the assay broadly applicable.
This assay is a convenient method to investigate the effects of the cell-matrix and cell-cell interaction on cell migration under different experimental conditions.
The first step of the assay is to culture a confluent cell monolayer which represents the in vivo conditions of the tissue before wounding. After the wound is created, cell migration and invasion are observed with real time visualization (Incucyte live cell imaging) until wound closure is reached which enables assessment of wound closure rates and morphological changes to cells.
Technique used by Pharmatest is designed to generate accurate and reproducible analysis results for wound healing assays, both for phase-contrast and fluorescence images.