How to Study Protein Recruitment to DNA Lesions by a Combination of UV Laser and White Light Laser

Cell cultivation

Human osteosarcoma U2OS cells stably expressing GFP-BMI1 originated from the laboratory of Prof. Maarten Van Lohuizen, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, The Netherlands. We obtained these cells from the laboratory of Assoc. Prof. Dušan Cmarko, Charles University in Prague. Mouse 3T3 cells were a generous gift from Dr. Paul Verbruggen from Swammerdam Institute for Life Sciences University of Amsterdam, The Netherlands. U2OS and 3T3 cells were cultivated in D-MEM medium (Dulbecco’s Modified Eagle Medium, PAN Biotech, GmbH, Germany) containing 10% fetal calf serum. Cultivation proceeded at 37 °C in a humidified atmosphere containing 5 % CO2.

Induction of DNA double stand breaks

U2OS cells stably expressing GFP-BMI1 and 3T3 cells stably expressing EGFP-HP1b were cultivated under standard conditions. For micro-­irradiation, the cells were sensitized with 10 μM 5-bromo-2’-deoxy-uridine (BrdU), 16–18 h before local irradiation. Control and TSA-treated U2OS and 3T3 cells were stained using BrdU Labeling (Roche, #11296736001). BrdU-sensitized cells were irradiated by UV laser (355 nm). We irradiated half of the nuclei or defined strips by 80% laser output, not reduced at acousto-optic tunable filter (AOTF). The following settings were used: format 512 × 512 pixels, 400 Hz, bidirectional mode, 64 lines, zoom >5–10x. After irradiation, the cells were fixed in 4% paraformaldehyde, and phosphorylated histone H2A.X (gH2AX) was detected by immunofluorescence methods with rabbit polyclonal antibody against gH2A.X (phospho S139; Abcam, #ab2893). For visualization of locally micro-irradiated live cells and fixed cells after immunofluorescence, we used confocal microscopy. These analyses were performed using white light laser (WLL, 470–670 nm in 1 nm increments) at 554 nm connected to the confocal microscope Leica TCS SP5 X. For visualization of biological objects we used a magnification of 64× and numerical aperture N.A. = 1.4.

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