Cytotoxicity, Apoptosis and Cell Death
Cytotoxicity Apoptosis and Cell Death
Last Updated on Saturday, 18 February 2012 19:05
Overview
Different cell death mechanisms, including necrosis, apoptosis and autophagy, are important cellular states and/or actions, with particular distinct characteristic responses and phenotypes. Some key markers to assess these mechanisms include the generation of reactive oxygen species, disruption of mitochondrial transmembrane potential, increased lysosomal mass and cell permeability, activation of caspases, alterations in nuclear morphology, and reduction in cell number. High-content is ideally suited to enable multiplexed measurements of combinations of these early toxicity markers.as well as provide the ability to monitor different forms of cell death such as necrosis, apoptosis and autophagy.
In vitro Toxicity Testing
A common goal of in vitro cytotoxicity testing is to determine the concentrations at which a compound causes toxicity. Since cytotoxicity is a multifactorial process, in vitro assays that can simultaneously monitor several independent indicators of toxicity in the cell will better detect the earliest onset of toxic effects by compounds. Tracking multiple independent toxicity indicators increases the predictive power of the assay and this is a "sweet-spot" for high-content
Apoptosis
The mitochondrial pathway of apoptosis is characterized by early events such as cytochrome c release from mitochondria and caspase 9 activation (cleavage). Activated caspase 9 cleaves and activates caspase 3 which then leads to cleavage of PARP in the nucleus. Other nuclear events (e.g., DNA fragmentation and nuclear condensation) occur at the same time.
Autophagy and Cell Death
Typically, small proteins are usually ubiquitinated and targeted for degradation, but the accumulation of large protein aggregates may lead to autophagy. Autophagy, a form of programmed cell death, is induced in a caspase-independent fashion. Autophagy is a catabolic process characterized by self-ingestion of cellular components such as organelles and long-lived proteins. Presence of LC3B on autophagosomes is a good marker of autophagy.
Application Example
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A common goal of in vitro cytotoxicity testing is to determine the concentrations at which a compound causes toxicity. Since cytotoxicity is a multifactorial process, in vitro assays that can simultaneously monitor several independent indicators of toxicity in the cell will better detect the earliest onset of toxic effects by compounds. Tracking multiple independent toxicity indicators increases the predictive power of the assay and this is a "sweet-spot" for high-content.
After toxic insult, cell death often results by either apoptosis or necrosis that are marked by numerous cellular events that can be monitored. Early, reversible events include cell cycle arrest, loss of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Later, irreversible events include change in nuclear morphology, nuclear condensation, DNA loss (pyknosis), nuclear fragmentation, loss of plasma membrane integrity, increased cell permeability and eventual cell loss.
The Thermo Scientific ArrayScan VTI HCS Reader, Cellomics HCS Reagents and Cell Health Profiling BioApplications enables simultaneous measurement of six independent parameters that monitor cell health, including cell loss, nuclear size and morphology changes, DNA content, changes in cell membrane integrity and cell permeability, mitochondrial membrane potential changes, and cytochrome c localization and release from mitochondria. The Reagents use Hoechst dye to monitor cell loss, nuclear morphology changes and DNA content whereas the other three parameters are monitored by separate fluorescent probes. |
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Assays and Reagents
Thermo Scientific Cellomics HCS Reagents and Assays are available for a wide variety of targets. Each assay offers all the components necessary to perform the assay as well as a validated protocol, with step-by-step instructions for use on any of the Thermo Scientific HCS Readers as well as other instrumentation and standard fluorescence microscopes. Reagents and Assays are available in kit form (everything in the box) or as components where based on your need, so you may purchase all or al-la-carte reagents for the assay. To order components for a legacy kit please visit our ReagentFinder page.
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Assay name |
Target/Detection Colors |
Product # |
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Apoptosis 1 Assay |
F-actin (green), mitochondrial membrane potential (red) and cell number and nuclear morphology (blue) |
K0400011 R0105121 |
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Autophagy: LC3B |
LC3B (orange) and cell number and nuclear morphology (blue) |
8407602 |
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Autophagy: LC3B and Poly-Ubiquitin |
LC3B (green), Poly-Ubiquitin (orange) and cell number and nuclear morphology (blue) |
8407802 |
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Caspase 3 Activation Assay |
Cleaved Caspase 3 (orange) and cell number and nuclear morphology (blue) |
8402202 |
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Caspase 9 Activation Assay |
Cleaved Caspase 9 (orange) and cell number and nuclear morphology (blue) |
8402302 |
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Cell Death Detection Assay |
LC3B (red), Cytochrome c (orange), cell permeability (green) and DNA content, cell number and nuclear morphology (blue) |
8408002 |
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Cell Viability Assay |
Dead cell (orange), live cell (green) and cell number and nuclear morphology (blue) |
K0200011 R0200021 |
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Cleaved PARP Detection Assay |
Cleaved PARP (orange) and cell number and nuclear morphology (blue) |
8402702 |
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Cytochrome C Detection Assay |
Cytochrome c (orange) and cell number and nuclear morphology (blue) |
8405602 |
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Cytotoxicity 1 Assay |
Membrane permeability (green), lysosome mass/pH (orange) and cell number and nuclear morphology (blue) |
K0200021 R0200061 |
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Cytotoxicity 2 Assay: Lysosomal and Mitochondrial Probes |
Membrane permeability (green), mitochondrial membrane potential (orange), lysosome mass/pH (orange) and cell number and nuclear morphology (blue) |
8400002 |
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Cytotoxicity 2 Assay: Lysosomal Probe |
Membrane permeability (green), lysosome mass/pH (orange) and cell number and nuclear morphology (blue) |
8400102 |
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Cytotoxicity 2 Assay: Mitochondrial Probe |
Membrane permeability (green), mitochondrial membrane potential (orange) and cell number and nuclear morphology (blue) |
8400202 |
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Cytotoxicity 3 Assay |
Cytochrome c (red), mitochondrial membrane potential (orange), cell permeability (green) and cell number and nuclear morphology (blue) |
8408102 |
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Poly-Ubiquitin Detection Assay |
Poly-Ubiquitin (orange) and cell number and nuclear morphology (blue) |
8407702 |
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Mitosis-Apoptosis Assay |
BrdU (green), DNA content, cell number and nuclear morphology (blue), p53 (red), caspase 3 (orange) |
8408202 8408203 |
