DNA Fragmentation Assay

Name: ChemoMetec
Price range: $

For Late Apoptosis Evaluation Using the NucleoCounter^® NC-3000™

Easily measure DNA fragmentation levels
Acquisition and analysis in one simple step
User-friendly protocol with predefined settings
No RNase treatment required
No calibration required
Clear data presentation with Plot Manager feature
Automated PDF reports
Export data in FCS/ACS formats

Overview of the DNA Fragmentation Assay

During apoptosis, proteins act on chromosomal DNA to fragment the DNA into ladders. This late-stage apoptotic event is detected by measuring DNA content in a way similar to that done in our Cell Cycle Assay. In apoptotic cells you will see a dissolution of peaks at the G1 and G2 phases, and the rise of a “sub G1 peak” which grows with the progression of the chromosomal DNA laddering.

The mechanism of the DNA degradation is the activation of calcium- and magnesium-dependent nucleases as apoptosis progresses. As a result, within the DNA there are nicks and double-strand breaks causing fragmentation¹. This is a late-stage apoptotic event, which can be detected by quantifying the cells’ DNA content. Late-stage apoptotic cells will have less DNA content and can thereby be identified as being in the sub-G1 phase.

Assay Principle

Using fluorescence microscopy and image analysis, the NucleoCounter^® NC-3000™ automatically detects cells with fragmented DNA (sub-G1 cells).

After staining fixed cells with DAPI, the sample is analyzed using the NucleoCounter^® NC-3000™ system, where cellular fluorescence is quantified and apoptotic cells with fragmented DNA are identified as a sub-G1 peak in a DNA content histogram displayed on the computer screen.

Markers in the histogram can be used to demarcate apoptotic cells.

Results Presented in Plot Manager

Jurkat cells were grown in the absence (upper row) or in the presence (lower row) of camptothecin and cells were analyzed using the DNA Fragmentation Assay and a NucleoCounter® NC-3000™.

Scatter plots and histograms were obtained using the NucleoView™ NC-3000™ software. Markers in the displayed histograms were used to demarcate cells with fragmented DNA (sub-G1 cells). The colored histograms are a merge of untreated (blue line) and camptothecin treated (red line) samples.

References

BD Larsen and CS Sørensen: The caspase‐activated DNase: apoptosis and beyond. FEBS J. 2017; 284(8): 1160-1170.

DNA Fragmentation Assay

For Late Apoptosis Evaluation Using the NucleoCounter® NC-3000™

Overview of the DNA Fragmentation Assay

Assay Principle

Results Presented in Plot Manager

References

Podcast: Standardizing Cell Counting

Consistency is King!

Cell Cycle Assay

Cell Assays

Oncology

Cell Biology

Apoptosis in Colorectal & Ovarian Cancer

Targeted Cancer Diagnoses & Therapies

Cell Counting in GMP Environments

Instrument Solutions

Tech notes

Tech Note: Variations and Statistics for NucleoCounter® NC-3000™

White Paper: NucleoView™ NC-3000™ Software and CFR 21 Part 11

Tect Note: LED Life Time Consideration for NucleoCounter® NC-3000™

Publications

MIM1, the Mcl-1 – specific BH3 mimetic induces apoptosis in human U87MG glioblastoma cells

Cytotoxic and proapoptotic effect of doxycycline – An in vitro study on the human skin melanoma cells.

Evaluation of cytotoxicity, genotoxicity, and apoptosis of wastewater before and afterdisinfection with performic acid.

Xylazine as a drug of abuse and its effects on the generation of reactive species and DNA damage on humanumbilical vein endothelial cells.

The role of UVA radiation in ketoprofen-mediated BRAF-mutant amelanotic melanoma cells death – A study at the cellular and molecular level

The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma

MIM1 induces COLO829 melanoma cell death through mitochondrial membrane breakdown, GSH depletion, and DNA damage

Mcl-1 Inhibitor Induces Cells Death in BRAF-Mutant Amelanotic Melanoma Trough GSH Depletion, DNA Damage and Cell Cycle Assay Changes

The Role of MITF and Mcl-1 Proteins in the Antiproliferative and Proapoptotic Effect of Ciprofloxacin in Amelanotic Melanoma Cells: In Silico and in Vitro Study

A Synthetic Chalcone Derivative, 2-hydroxy-3′,5,5′-trimethoxychalcone (DK-139), Triggers Reactive Oxygen Species-Induced Apoptosis Independently of p53 in A549 Lung Cancer Cells

Moxifloxacin as an Inducer of Apoptosis in Melanoma Cells: A Study at the Cellular and Molecular Level

Exploring Mitochondria-Mediated Intrinsic Apoptosis by New Phytochemical Entities: An Explicit Observation of Cytochrome c Dynamics on Lung and Melanoma Cancer Cells

Ceragenin CSA-13 as Free Molecules and Attached to Magnetic Nanoparticle Surfaces Induce Caspase-Dependent Apoptosis in Human Breast Cancer Cells via Disruption of Cell Oxidative Balance

BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma

The Synthetic Chalcone Derivative 2-hydroxy-3′,5,5′-trimethoxychalcone Induces Unfolded Protein Response-Mediated Apoptosis in A549 Lung Cancer Cells

Blockade of the Ras/Raf/ERK and Ras/PI3K/Akt Pathways by Monacolin K Reduces the Expression of GLO1 andInduces Apoptosis in U937 Cells

Deubiquitinating activity of CYLD is impaired by SUMOylation in neuroblastoma cells.

Differentiation and apoptosis induction by lovastatin and γ-tocotrienol in HL-60 cells via Ras/ERK/NF-κB andRas/Akt/NF-κB signaling dependent down-regulation of glyoxalase 1 and HMG-CoA reductase

C5-curcuminoid-4-aminoquinoline based molecular hybrids: design, synthesis and mechanistic investigation of anticancer activity

Polish natural bee honeys are anti-proliferative and anti-metastatic agents in human glioblastoma multiformeU87MG cell line.

CYLD controls c-MYC expression through the JNK-dependent signaling pathway in hepatocellular carcinoma

Imidazolium-derived ionic salts induce inhibition of cancerous cell growth through apoptosis

Biological Activity of N-Hydroxyethyl-4-aza- 2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells

Anticancer activity of 4-aminoquinoline-triazine based molecular hybrids

Ultraviolet C Irradiation Induces Different Expression of Cyclooxygenase 2 in NIH 3T3 Cells and A431 Cells: The Roles of COX-2 Are Different in Various Cell Lines

Novel Nitrobenzazolo[3,2a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Assay Changes, and Mitrochondrial Permeabilization

Expression of Id proteins is regulated by the Bcl-3 proto-oncogene in prostate cancer

Effects of zinc supplementation and zinc chelation on in vitro β-cell function in INS-1E cells

Cobalamin Deficiency: Effect on Homeostasis of Cultured Human Astrocytes

Extracellular vesicles released by fibroblasts undergoing H-Ras induced senescence show changes in lipid profile

Investigation of the cytotoxic, genotoxic, and apoptosis-inducing effects of estragole isolated from fennel(Foeniculum vulgare).

Association of nuclear-localized Nemo-like kinase with heat-shock protein 27 inhibits apoptosis in human breastcancer cells.

Application notes

App Note DNA Fragmentation NC-3000™

For Late Apoptosis Evaluation Using the NucleoCounter^® NC-3000™

Apoptosis in
Colorectal &
Ovarian Cancer

Targeted Cancer Diagnoses
& Therapies

Cell Counting in
GMP Environments