Cell health and viability measurements provide essential insight into a broad range of biological processes and treatment responses. Diverse assay formats and reagents have been developed that measure specific aspects of cell viability corresponding to particular cellular response pathways and mechanisms of injury. Membrane impermeable fluorometric dyes, or apoptotic and necrotic specific markers, can be used in conjunction with label-free total cell counts to provide relative measures of cell health within a population. Activation of cellular stress pathways can be detected using expressed biosensors and detailed kinetic imaging, revealing subtle treatment-induced effects, while the comet assay and γ-H2AX immunofluorescence assay enable characterization of genotoxic-specific effects. Agilent BioTek instrumentation and Gen5 software provide automated solutions for a full spectrum of cell health and viability applications.
Genotoxicity refers to the destructive effects of mutagens such as high energy radiation and chemicals on cellular nucleic acids, particularly nuclear DNA. Single cell gel electrophoresis, or the comet assay, can be used to probe DNA fracture by mutagens and the γ-H2AX immunofluorescence assay can used to quantify DNA double strand breaks. Each method has its challenges with respect to image analysis, but Agilent BioTek Gen5 data analysis software 3.0 has the ability to quantify either the %DNA in the comet tail or comet tail moment; or count γ-H2AX foci to determine levels of genotoxicity.
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Apoptosis, or programmed cell death, refers to a series of biochemical changes in the cell leading to distinct morphological changes that can be assessed using fluorescent probes or contrast-enhancing microscopy modes such as phase contrast. Examples of fluorescent probes include those that target phosphatidylserine which becomes exposed to extracellular reagents in early stage apoptosis (i.e. pSIVA-IANBD), fluorometric substrates of or antibodies to caspases (i.e. Caspase 3/7) that generate fluorescence with caspase activation, or probes that measure chromatin condensation. Agilent BioTek Cytation and Agilent BioTek Lionheart FX both have the ability to measure apoptosis through the use of fluorescent probes or by label-free methods relying on phase contrast microscopy.
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The ability to monitor cell stress and toxicity and its resulting effects on cellular signaling is important for both understanding disease pathology and in the drug discovery and development processes. The reversible cell stress biosensor from Montana Molecular provides a sensitive method for detecting cell stress and toxicity within a population.
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