Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry.
Nadia Vezzio-ViéMarie-Alice Kong-HapEve CombèsAugusto Faria AndradeMaguy Del RioPhilippe PaseroCharles TheilletCéline GongoraPhilippe PourquierPublished in: International journal of molecular sciences (2022)
The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo ® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.
Keyphrases
- dna damage response
- single cell
- dna repair
- dna damage
- cell cycle
- flow cytometry
- induced apoptosis
- circulating tumor
- high resolution
- cell free
- single molecule
- cell therapy
- oxidative stress
- signaling pathway
- cell proliferation
- deep learning
- randomized controlled trial
- clinical trial
- south africa
- stem cells
- epithelial mesenchymal transition
- pi k akt
- cell death
- drug induced
- circulating tumor cells