Assessing DNA Damage Foci: To Identify Radiation-Induced DNA Damage Foci in Cancer Cell Line Using Immunofluorescence Staining

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

1. 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.
2. Grow cells in a humidified 5% CO₂ environment at 37 °C until 70% confluency with medium renewal every 2 to 3 days.
3. To gain BNCT beam (e.g., neutron beam plus BPA) and BNCT effect of killing cancer cells as heavy particles and to deposit most of their ener....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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