Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining
Published: April 30, 2023
Abstract
Source: Maliszewska-Olejniczak et al. Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells. J. Vis. Exp. (2020).
This video describes a protocol to detect radiation-induced DNA repair foci in the colon cancer cell lines using immunofluorescence staining. DNA repair proteins get activated at the DNA damage site; therefore, we can detect them using specific antibodies and visualize them under a fluorescence microscope.
Protocol
1. Preparation of cell culture and experimental set-up Maintain human colon cancer cells, HCT-116 as recommended by the supplier, in monolayers in 75 cm2 culture flasks containing 10 mL of formulated McCoy's 5a Medium Modified, supplemented with 10% fetal bovine serum and 1% antibiotic antimycotic solution. Grow cells in a humidified 5% CO2 environment at 37 °C until 70% confluency with medium renewal every 2 to 3 days. …
Disclosures
The authors have nothing to disclose.
Materials
| 12 mm Coverslips | VWR | 89015-725 | |
| 35 mm Petri dishes | Sarstedt | 7.183.390.000 | |
| 4-Borono-L-phenylalanine | SIGMA-ALDRICH | 17755 | |
| Antibiotic-Antimycotic (100X) | Gibco | 15240062 | |
| Anti-DNA PKcs (phospho S2056) antibody – ChIP Grade | Abcam | AB18192 | |
| Anti-Mouse IgG (whole molecule)– FITC antibody produced in goat | SIGMA-ALDRICH | F0257 | |
| Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 | MerckMillipore | 05-636 | |
| Anti-RAD52 antibody | Abcam | AB117097 | |
| Bovine Serum Albumin Fraction V (BSA) | Roche | BSAV-RO | |
| DAPI (4',6-Diamidino-2- Phenylindole, Dihydrochloride) | ThermoFisher SCIENTIFIC | D1306 | |
| Fetal Bovine Serum (Heat Inactivated) | SIGMA-ALDRICH | F9665 | |
| Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) | Abcam | AB150077 | |
| HCT-116 cell line | ATCC | CCL-247™ | |
| ImageJ | National Institute of Health (NIH) | https://imagej.nih.gov/ij/ | |
| Image Pro | Media cybernetics | https://www.mediacy.com/imagepro | |
| microscope slides | ThermoFisher SCIENTIFIC | B-1198 | |
| Phosphate Buffered Saline (PBS) | Hirszfeld Institute of Immunology and Experimental Therapy, PAS | 20.59.52.0 | |
| Triton X-100 | SIGMA-ALDRICH | X100 | |
| Trypan Blue Stain, 0.4% | Logos Biosystems | T13001 | |
| Trypsin-EDTA solution 0.25% | SIGMA-ALDRICH | T4049 |
Cite This Article
Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining. J. Vis. Exp. (Pending Publication), e20325, doi: (2023).