Quantifying DNA damage is mandatory to assess (i) potential adverse effects of candidate drugs, molecules or extracts developed in the dermo-cosmetic industry, and (ii) the efficacy of anticancer therapies, in order to induce tumor cell genotoxicity. Genotoxic potential effect can be analyzed by different assays, like

gamma-H2AXComet assay  – Micronucleus and dual H3/H2AX assay

HCS Pharma developed and validated these different assays in an automated fashion on HepG2 cells. They can also be performed on primary cells and other cell lines.

To learn more on these assays, please refer to our posters that have been presented in different congresses:

gH2AX detection and colocalisation with 53BP1

Study of DNA integrity

DNA double-strand breaks (DSBs) may be encountered in response to exogenous stimuli; they are triggered either by ionizing radiations or DNA-damaging chemicals. In response to DSBs, H2AX is phosphorylated within minutes at Ser 139, and is then referred to as γH2AX. This assay can be multiplexed to look for 53BP1/γH2AX colocalization.

Requirements

  • Compounds: powder or stock solution
  • Cells: adherent cells, primary cells or cell lines, with or without S9 if required
  • Plate format: 96 or 384-well plates

Example of Etoposide

HepG2 cells were exposed 3 hours to increasing concentrations of etoposide

Don’t hesitate to ask for our short report on HepG2 cells exposed to our in-house genotoxicity library !

 

Micronucleus and dual H3/H2AX assay

Clastogen or Aneugen ? That is the question

Distinguishing aneugenic (chromosomes loss) from clastogenic effects (DNA damages) is essential to assess genotoxic properties of compounds.

  • In response to a clastogen DNA double strand breaks (DSBs), H2AX is phosphorylated within minutes at Ser 139, and is then referred to as γH2AX. Detection of γH2AX spots allows to determine specific DSB unrelated to unspecific phosphorylation (e.g. apoptosis).
  • Contrary to γH2AX, micronuclei are a late marker of damages to the DNA and can be related to aneugenic and clastogenic effects.
  • Histone H3 is phosphorylated during mitosis and is referred as γH3. Aneugenic compounds induce phosphorylation of H3 due to mitosis arrest, and confirm micronuclei detection.

By detecting both dual γH2AX/γH3 staining and micronuclei on our high content screening platform, our assay provides an efficient tool to assess the genotoxic properties of your compounds.

Requirements

  • Compounds: powder or stock solution
  • Cells: adherent cells, primary cells or cell lines, with or without S9 if required
  • Plate format : 96 or 384-well plates

Resultats and test robustness

This assay is validated on HepG2 cells exposed 24 hours to compounds from our in-house genotoxicity library. Cell line stability was assess on 4 passages and experiments were performed thought a biological triplicate.

We detected 100% and of the clastogen tested. And 75% of the aneugen tested were statistically positive for micronucleus detection.

Exemple of BAP : HepG2 cells were exposed 24 hours to increasing concentrations of Benzo[a]pyrene (B[A]P).

Don’t hesitate to ask for our short report on HepG2 cells exposed to our in-house genotoxicity library !

 

Automated Comet assay

Study DNA integrity

The comet assay is a sensitive, well-established technique for quantifying DNA damage in eukaryotic cells. Compatible with the detection of a wide range of DNA damaging agents, its consists in making fragmented DNA migrate in an electrophoresis gel; while damaged DNA forms the tail of the comet, intact DNA moves at a slower rate and forms the head of the comet. The percentage of fragmented DNA in the comet provides a direct measurement of DNA damage.

Requirements

  • Compounds: powder or stock solution
  • Cells: suspension or adherent, primary cells or cell line, with or without S9
  • Trevigen automated 96-well comet assay

Example of Etoposide

HepG2 cells were exposed for 3 hours to increasing concentrations of Etoposide.

Don’t hesitate to ask for our short report on HepG2 cells exposed to our in-house genotoxicity library !

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