GFP-NBS1, GFP-EXO1, GFP-Ku70, GFP-XLF, or GFP-XRCC4 were transfected into HCT116 DNA-PKcs +/+, −/−, or KD/− cells with JetPrime® (Polyplus) following the manufacturer's instructions. Twenty-four hours after the transfection laser micro-irradiation and real-time recruitment was performed with a Carl Zeiss Axiovert 200M microscope with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss) as previously described (28 (link)). DSBs were generated with a 365-nm pulsed nitrogen laser (Spectra-Physics), which was directly coupled to the epifluorescence path of the microscope (28 (link)). Time-lapse images were taken via a Carl Zeiss AxioCam HRm camera. The cells were maintained in a CO2-independent medium (Invitrogen) at 37°C during micro-irradiation and time-lapse imaging. Fluorescence intensities of the micro-irradiated area and control area were determined by Carl Zeiss Axiovision software, v4.5, and the intensity of irradiated was normalized to non-irradiated control area as previously described (26 (link)).
365 nm pulsed nitrogen laser
Manufactured by Spectra-Physics
The 365-nm pulsed nitrogen laser is a compact and reliable laser source that generates ultraviolet pulses at a wavelength of 365 nanometers. It is designed for a wide range of scientific and industrial applications that require a stable and consistent UV light source.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m microscope, by Zeiss (4 mentions)
Plan apochromat 63x na 1.40 oil immersion objective, by Zeiss (3 mentions)
Axiovision software version 4, by Zeiss (2 mentions)
Co2 independent medium, by Thermo Fisher Scientific (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 63 na 1.40 oil immersion objective, by Zeiss (1 mentions)
6 protocols using 365 nm pulsed nitrogen laser
1
Real-Time Recruitment of DNA Repair Factors
2
Laser Micro-Irradiation for Live-Cell Imaging
3
Immunostaining for DNA Damage Foci
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For immunostaining, cells were grown on coverslips and irradiated via the 365-nm pulsed nitrogen laser (Spectra-Physics) or 8 Gy of IR. For the cells irradiated with the laser, the coverslip was placed in a dark box and the cells were allowed to recover at 37°C for 30 min. The cells were subsequently washed with ice cold PBS three times and then fixed via incubation with 4% paraformaldehyde at room temperature for 20 min. The cells were permeablized with PBS containing 0.5% Triton X-100 on ice for 10 min, blocked with PBS containing 5% normal goat serum overnight at 4°C and then incubated with the primary antibodies at room temperature for 1 h. After washing with cold PBS, cells were incubated with the second antibodies conjugated with Alexa 488 (Invitrogen, A11001) or Texas red (Invitrogen, T2767). Following IR with 8 Gy, focus formation of RPA and Rad51 was detected using fluorescent immunostaining at various time points as described in the figure legends and as previously described (30 (link)).
4
Live-Cell Imaging and Laser Micro-Irradiation
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Live cell imaging combined with laser micro-irradiation was carried out as previously described (11 (link),29 (link)). Fluorescence was monitored via an Axiovert 200 M microscope (Carl Zeiss, Inc.), with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss, Inc.). A 365-nm pulsed nitrogen laser (Spectra-Physics) was directly coupled to the epifluorescence path of the microscope and used to generate DSBs in a defined area of the nucleus. For quantitative analyses, the same amount of DNA damage was generated under standardized micro-irradiation conditions (minimal laser output of 75% for 5 pulses) in each experiment. Time-lapse images were taken with an AxioCamHRm camera. The fluorescence intensities of micro-irradiated and non-irradiated areas within the cell nucleus were determined using the AxioVision Software, version 4.8 (Carl Zeiss, Inc.). Each data point is the average of 10 independent measurements.
5
Live Cell Imaging and Laser Micro-Irradiation
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Live cell imaging combined with laser micro-irradiation was performed as previously described (6 (link),7 (link)). Fluorescence was monitored via an Axiovert 200 M microscope (Carl Zeiss, Inc.), with a Plan-Apochromat 63×/NA 1.40 oil immersion objective (Carl Zeiss, Inc.). A 365-nm pulsed nitrogen laser (Spectra-Physics) was directly coupled to the epifluorescence path of the microscope and used to generate DSBs in a defined area of the nucleus. For quantitative analyses, the same amount of DNA damage was generated under standardized micro-irradiation conditions (minimal laser output of 75% for five pulses) in each experiment. Time-lapse images were taken with an AxioCamHRm camera. The fluorescence intensities of micro-irradiated and non-irradiated areas within the cell nucleus were determined using the AxioVision Software, version 4.8 (Carl Zeiss, Inc.). Each data point is the average of ≥10 independent measurements.
6
Laser-Induced DNA Double-Strand Breaks
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A 365‐nm pulsed nitrogen laser (Spectra‐Physics) was applied to generate DSBs in a defined area of the nucleus in cells. At 30 minutes after laser radiation, cells were fixed and subjected to immunofluorescence.
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