Live-Cell Imaging and Laser Micro-Irradiation

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.

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Lee K.J., Saha J., Sun J., Fattah K.R., Wang S.C., Jakob B., Chi L., Wang S.Y., Taucher-Scholz G., Davis A.J, & Chen D.J. (2015). Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase. Nucleic Acids Research, 44(4), 1732-1745.

Publication 2015

Axiovert 200 M microscope Plan-Apochromat 63X/NA 1.40 oil immersion objective 365-nm pulsed nitrogen laser AxioVision Software, version 4.8

Cell Cell nucleus Dna damage Dsbs Fluorescence Immersion M 200 Microscope Nitrogen laser Pulses

Corresponding Organization :

Other organizations : The University of Texas Southwestern Medical Center, GSI Helmholtz Centre for Heavy Ion Research

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Variable analysis

independent variables

Laser micro-irradiation conditions (minimal laser output of 75% for 5 pulses)

dependent variables

Fluorescence intensities of micro-irradiated and non-irradiated areas within the cell nucleus

control variables

Live cell imaging combined with laser micro-irradiation procedure as previously described (11, 29)
Axiovert 200 M microscope (Carl Zeiss, Inc.) with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss, Inc.)
365-nm pulsed nitrogen laser (Spectra-Physics) directly coupled to the epifluorescence path of the microscope
AxioCamHRm camera for time-lapse imaging
AxioVision Software, version 4.8 (Carl Zeiss, Inc.) for fluorescence intensity measurements

controls

Each data point is the average of 10 independent measurements

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