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

Overview

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 energy within the boron-containing cancer cells, the day before irradiation, add boron delivery agent to the cell culture medium – e.g., ¹⁰BPA, 4-Borono-L-phenylalanine (10 μg ¹⁰B/mL, 0.925 mM final) 12–16 h (maximum uptake of boron) before the irradiation.
   NOTE: If using another cell line, for which no data exists in the case of BNCT study, test the boron uptake for each cell line to obtain optimal boron concentration. Measure the BPA cellular uptake by inductively coupled plasma optical emission spectroscopy (ICP-OES) as performed by Dagrosa group.
4. After 12-16 h of boron delivery, aspirate the medium and wash the cells with 10 mL of 1x PBS. Then trypsinize the cells by adding 2 mL of Trypsin-EDTA solution to the cells and incubate for 5 min at 37 °C to enhance the cell detachment. When cells start to detach, stop the trypsin action by adding 10 mL of complete medium.
5. Aspirate the cell suspension in the 15 mL tube and count the cells using an automated cell counter by adding 10 µL of 0.4% trypan blue to 10 µL of cells, mix, and aliquoting 10 µL on a counting slide. Dilute the cell suspension to a concentration of 1 x 10⁶ cells/mL of the medium. Aliquot 1 mL of the cell suspension per cryotube.
6. Prepare thermoluminescent dosimeters (TLDs) for the measurement of gamma-ray doses and gold foils for neutron fluencies and attach TLDs to the cryotubes or cell culture flask before the irradiation.
7. Irradiate the 1 x 10⁶ cells/mL with the neutron-mixed beam at recommended minimal thermal neutron flux of 5.9 x 10¹¹ (n/cm^-2 min^-1)⁴ in culture flasks or cryotubes depending on the facility capabilities.
   NOTE: Set up the time of irradiation before to obtain recommended neutron flux.
8. After irradiation, seed 1 mL of irradiated cells (1 x 10⁶ cells/mL) on 22 x 22 mm coverslips in 35 mm Petri dishes or 6-well plates, supplement with 2 mL of required medium. Incubate cells for a maximum of 3 h at 37 °C in a humidified 5% CO₂ environment to let them attach to the coverslips. Observe the attachment of cells from time to time under an inverted microscope with 20x objective.
   NOTE: For HCT-116 cells, the minimum density is 600,000 cells to seed. For a quick further staining procedure, use chamber slides, reduce cell density accordingly.

2. Fixation of cells

1. After irradiation and incubation of cells, remove the medium from the attached cells and wash cells once with 2.5 mL of PBS.
2. Fix cells with 1 mL of 70% ethanol for 10 min at RT.
   NOTE: Alternatively, use 1%-3.7% paraformaldehyde (PFA) for fixation as recommended previously. The protocol is paused here. Store fixed cells in ethanol in a freezer at -20°C for not more than a few weeks for further analysis and immunofluorescence staining.

3. Permeabilization of cells

1. After fixation, remove ethanol from the Petri dish and wash with 2.5 mL of 1x PBS.
   NOTE: Do not let the cells dry between the rinsing steps.
2. Gently add 1 mL of 0.2% of Triton X-100/PBS to cover the coverslips with cells in Petri dishes.
3. Incubate for 5 min at RT.
4. Wash the cells 3x with 2.5 mL of 1x PBS.
   NOTE: Increase the percentage of Triton X-100 in PBS up to 0.5% if needed (more foci detectable, dependent on the tested cell line). Perform additional washes with PBST (PBS with 0.5% Tween) to avoid unspecific binding.
5. Block permeabilization step with 1 mL of 2% BSA (Bovine Serum Fraction V albumin) diluted in PBS and incubate for a minimum of 30 min or 1 h with 1% BSA.
   NOTE: This step can be omitted and is dependent on the type of antibody used. Alternatively, use 5% FBS as recommended.

4. Immunofluorescence staining

1. Add the proper amount of primary antibody (Anti-ɣ-H2AX, Anti-DNA-PKcs, and Anti-Rad52) diluted in PBS with BSA (2% Bovine Serum Fraction V albumin) as recommended (see Table 1 with recommended dilutions) (100 µL is needed per sample).

Primary antibody	Function	Recommended dilution	Storage [°C]
Anti-ɣ-H2AX	detection of DNA-DSBs	1:1000 (PBS-BSA)	4
Anti-DNA-PKcs	detection of RIFs of DNA-PKcs protein belonging to NHEJ	1:200 (PBS-BSA)	-20
Anti-Rad52	detection of RIFs of Rad52 protein belonging to HRR	1:200 (PBS-BSA)	-20
Secondary antibody		in the dark
anti-mouse IgG FITC	ɣ-H2AX foci	1:400 (PBS-BSA)	4
Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)	for NHEJ and HRR repair proteins foci	1:500 (PBS-BSA)	-20

Table 1: Recommended dilutions and notes for the usage of antibodies used in section

2. Cover the Petri dish to maintain the humidity in a plastic box with moisturized lignin using distilled water and incubate for 30 min at 37 °C in the incubator.
   NOTE: The protocol can be paused here. Incubation can be performed at 4 °C overnight.
3. After incubation, perform 3 washes with 2.5 mL of PBS.
   NOTE: Do not allow the slide to dry during rinsing steps.
4. Add the proper amount of secondary antibody (anti-mouse IgG FITC, Goat Anti-Rabbit IgG (Alexa Fluor 488) diluted in PBS with BSA (see Table 1) (100 µL is needed per sample). For this and the following steps, work in the dark.
5. Cover the Petri dish again to maintain the humidity in a plastic box with moisturized lignin and incubate for a minimum 30 min at 37 °C in the incubator.
6. After incubation, perform 3 washes with 2.5 mL of PBS.
7. Add 100 µL of DAPI diluted in PBS to a final concentration of 1 µg/mL to counterstain the nuclei.
8. Incubate shortly for a maximum of 2 min at RT.
9. After incubation, perform 3 washes with 2.5 mL of PBS.
10. Remove the PBS and gently put a coverslip on the top of the mounting medium, avoiding the formation of air bubbles, and seal edges of the coverslip with nail polish. Wait until the varnish dries and paint the coverslip around.
11. Wait for the hardening of the mounting medium (up to 3 h) before image analysis under fluorescence microscopy.
    NOTE: The protocol can be paused here. Store glass slides in a plastic box at 4 °C in the dark.

Materials

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