DNA damage

Summary

DNA damage

Principle

Genomic DNA is susceptible to damage by both endogenous and exogenous factors. Laser microradiation across the nucleus rapidly produces localized chromatin-associated DNA damage, and laser microradiation can induce several types of DNA damage, including double-strand breaks (DSBs), single-strand breaks (SSBs), and oxidized bases.

Operation method

Laser microradiation-induced DNA damage

Principle

Genomic DNA is susceptible to damage by both endogenous and exogenous factors. Laser microradiation across the nucleus rapidly produces localized chromatin-associated DNA damage, and laser microradiation can induce several types of DNA damage, including double-strand breaks (DSBs), single-strand breaks (SSBs), and oxidized bases.

Materials and Instruments

Live cell microscope compatible petri dishes or culture dishes, adherent cell lines, CO
2
control, 5′-iodo-2-deoxyuridine (IdU), Hoechst 33342, 4% formaldehyde, PBS, containment solution, Triton X-100, mouse anti-γH2AX, mouse IgG (H + L) secondary antibody, bovine serum albumin (BSA), Tween 20, cell culture incubator; in addition, the confocal microscope for this experiment should be equipped with at least two lasers. In addition, the confocal microscope should be equipped with at least two lasers for this experiment, a 355 nm or 405 nm laser for micro-irradiation and another (e.g., 488 or 560 nm) for imaging. The microscope should be equipped with an incubation chamber for controlling temperature, CO
2
and humidity.

Move

1. Cell culture: 2-3 days prior to the start of the microirradiation experiment, cells are inoculated in Petri dishes and grown directly on 35 mm round imaging dishes. (Typically, 0.5~1 × ¹⁰⁶ cells are sufficient for most multifactorial studies)

2. Pretreatment with a photosensitizer, such as a halogenated nucleotide analog [bromodeoxyuridine (BrdU), 5′-iodo-2-deoxyuridine (IdU)] or a dye that binds to the DNA grooves of the cells being examined (Hoechst 33258, Hoechst 33342) is required prior to irradiation to allow for the energy uptake required to induce DSB. Incubate the cells with 10 μM bromodeoxyuridine (BrdU) for at least 24 hours or sensitize the cells with 10 μg/ml Hoechst 33342 for 10 minutes, and they will be ready for use approximately 60 minutes after the addition of the Hoechst.

3. Add pre-warmed _{CO2-conditioned} live cell medium supplemented with 10% FBS and phenol red free antibiotics to the cells to minimize autofluorescence. 4.

4. Microradiation Setup: Select the radiation laser (355 nm or 405 nm) to be used for DNA damage induction and set the exposure time in ms (for the 355 nm laser, create an exposure time of SSB (150 ms total), or DSB (250 ms total) for DNA damage. This will result in approximately 6 to 12 (for DSB) or 5 to 8 (for SSB) line/frame (if other shapes are used) scans, respectively, to induce DNA damage. Typically, for a 60× objective, a straight line will produce damage of 0.2 to 0.5 μm in width over the length of the line.

5. Live cell imaging: A series of confocal images of a mid-Z section is recorded at 2-second intervals before and after microirradiation. 6.

6. Immunodetection of desired proteins

(1) Remove the cell culture chamber from the microscope and incubate the cells at 37 °C in a humid atmosphere containing 5% CO2 for 5-10 minutes. After incubation, cells were washed with 0.5 mL of PBS (137 mM NaCl, 2.7 mM KCl, 8 mM _Na2HPO4, and 2 mM _KH2PO4 ) and fixed in 0.5 mL of 4% formaldehyde in PBS for 10 minutes at room temperature;

(2) Cells were washed once with PBS and then with 50 mM _NH4Cl to remove the residual formaldehyde;

(3) The cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min;

(4) Incubate the samples in a closed solution (5% FBS, 3% BSA, 0.05% Triton X-100 in PBS) for 40~60 min;

(5) Wash the cells once with PBS;

(6) Add primary antibody, e.g., mouse _anti-γH2 AX diluted in blocking buffer, and incubate overnight at 4 ℃;

(7) Wash the cells for 5 minutes in 3× containment solution;

(8) Add secondary antibody, e.g. donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor® 488 diluted in 4% BSA/PBS, and incubate for 1 hour at room temperature;

(9) Wash for 5 minutes in a 3× containment solution;

(10) Add fresh PBS and store at 4 °C.

Caveat

1. The researcher needs to consider that the laser parameters - wavelength, pulse length, pulse frequency, variable "zoom" of the microscope/laser system, exposure time, and energy - greatly influence the type and number of lesions induced by the beam, and thus the cellular response.

2. Special attention should be paid to the handling of Hoechst (a DNA insertion agent) and formaldehyde (moderately toxic by skin contact and inhalation), and work should be carried out in a well-ventilated area or fume hood.

Common Problems

(1) The cells look unhealthy, either because of the poor initial state of the cells or because of the intensity of the laser; excessive DNA damage can cause apoptosis;

(2) Cells move during image acquisition, cells change focus during image acquisition;

(3) Protein recruitment was not observed during laser microirradiation, change the laser parameters to reexperiment.

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