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Flow Cytometry Applications - Cell Health

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Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit.
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Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit showed that treatment with 2, 6-dimethoxyquinone results in decrease in mitochondrial potential, increased apoptosis and cell death under conditions evaluated.

Cell Health & Quality – Mitochondrial Analysis

Mitochondria are critical cellular organelles that produce 90% of cellular energy, control cell survival by regulating apoptosis, and produce reactive oxygen species (ROS). Mitochondrial superoxide generation results in oxidative stress, damage and cell death by apoptosis or to cellular energetic decline. Therefore, mitochondrial dysfunction caused by disease or compound treatment has dire consequences that can result in cell death. Monitoring the impact on mitochondria and related cell health markers is an important part of drug screening programs, pathway mapping, apoptosis, and disease research.

Flow cytometry detects multiple markers simultaneously at various stages of apoptosis, making it a powerful technique for studying pathways governing cell health and cell death. A variety of FlowCellect™ Kits can now be employed to assess changes in mitochondrial membrane potential, apoptosis as measured by Annexin V binding, mitochondrial oxidative stress, and cell death.

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Download our newsletter containing an artlcle on mitochondrial analysis HERE.

Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit.
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DNA Damage

Evidence suggests there is a direct correlation between DNA damage and cell cycle. Cells that are defective in DNA damage pathways can cause cancer because they lack the ability to sense and repair the damage, leading to genetic instability and ultimately uncontrolled cell growth.

The main kinase activated in response to double-stranded DNA breaks is ATM or Ataxia telangiectasia mutated kinase. ATM is a member of the phospho inositide 3-kinase (PI3K)-related Ser/Thr protein kinase family. Inactive ATM exists as a dimer but quickly dissociates and becomes phosphorylated on Serine 1981 in response to ionizing radiation. Once activated, ATM phosphorylates a number of downstream factors, including P53, CHK2, SMC1, NBS1, and Histone H2A.X. Amnis® imaging flow cytometers can be used to quantitate gamma-H2AX foci.

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Download our newsletter containing an artlcle on DNA damage HERE.

Live versus dead discrimination as a simple evaluation of cytotoxicity
Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit.
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Cell Toxicity

The guava® CellToxicity reagent uses a simple, non-radioactive protocol for measurement of cell cytotoxicity by a combination of carboxyfluorescein diacetate succinimidyl ester (CFSE) and 7-aminioactinomycin D (7-AAD). The kit can be used to measure effector and target cell health.

Applications for guava® Cell Toxicity Reagent:

  • Cell-mediated or Cell dependent cytotoxicity (CMC or CDC)
  • Antibody-dependent cell mediated cytotoxicity (ADCC)
  • Natural Killer Cytotoxicity (NK)

Download our application note on cell toxicity HERE.

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Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit.
Track RNA processing in single cells using our flow-validated conjugated antibody for Y14, an RNA-binding protein. Here, HeLa cells were stained with either MilliMark™ anti-Y14-FITC, clone 4C4 (green) or with IgG2b isotype control antibody (grey) and analyzed using flow cytometry

Epigenetics & Gene Regulation

A variety of kits and reagents can be used to evaluate epigenetics and gene regulation by flow cytometry. For example, all of the products located under “Cell Health & Quality – DNA Damage can help identify agents which induce DNA damage. Moreover, MilliMark™ reagents for epigenetics and gene regulation, such as the clone shown here, can also be used to track mechanisms of chromatin regulation, transactional and post-transcriptional regulation, translation and more in user-defined assays.

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Treatment of Jurkat cells with 2/6-dimethoxyquinone followed by analysis with the MitoDamage kit.

Autophagy

Autophagy is a catabolic process, evolutionarily conserved from yeast to mammals, involving the degradation of a cell's own components through formation of a distinct structure, the autophagosome.

Malfunctions of autophagy have been linked to human pathologies, including cancer and neurodegenerative disorders, and are now a major research area, particularly with respect to how tumor cells use autophagy to evade chemotherapy-induced cell death.

Download our application note on autophagy HERE.

Cell Death & Apoptosis

Knowing the performance profile of your cells prior to running your bioassay can mean the difference between valid assay results and wasted reagents, lost time and discarded data. Such analyses also aid in establishing uniform standards of cellular performance across long-term research studies, or across different sites.

Many applications can be performed with benchtop flow cytometers to evaluate indicators of cell health and performance, such as the apoptotic fraction and stage of apoptosis, transfection efficiency and viability, as well as cell counts.

Download our newsletter containing an artlcle on cell death & apoptosis HERE.

Evaluation of Apoptosis and Cell Death following compound treatment using the Guava® Nexin Assay
Cells were incubated for 4 hours in either control medium (DMSO or three test compounds camptothecin, 5-azacytidine and emetine (10mM final concentration). Following treatment, apoptotic progression was assessed using the Guava® Nexin Assay. Subpopulations are defined in the dot plots as healthy (teal), early (blue), or late-stage apoptotic (magenta) through combined annexin V and 7-AAD staining. Values represent the frequency of total apoptotic cells in a given sample.

Cell Cycle

Cell cycle phase distributions can be used to assess cell health, proliferation, as well as the potential mechanism of antineoplastic agents. For example, measuring the population of S phase cells can reflect the amount of newly synthesized DNA. Also, distinguishing cells in G2 from M phase cells can help identify cells undergoing mitosis. We offer a wide range of assays in this area for evaluations of cell cycle.

Download our application note on cell cycle HERE.

Detection of Cell Cycle Stages in PC3 cells following compound treatment
PC3 cells were incubated with: A) medium only (negative control); B) treated with emetine HCI; C) mechlorethamine; or D) albendazole for 24 hours after synchronization. Representative plots are shown here.

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