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

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).

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.