Glass bottom

The cells were seeded in the 2.5 mm culture dish with glass bottom (Cellvis) for 24 h and then transfected with plasmids containing fluorescent variants of the desired genes. After another 24 h, the transfected cells were observed under confocal laser scanning microscope FluoView FV1000 (Olympus Corporation) using 543 nm excitation for RFP fluorescence and 488 nm excitation for GFP and for differential interference contrast (DIC) observation. UPlanSAPO 60 × NA1.35 oil-immersion objective was used for imaging. For long-term monitoring, the culture dish was sealed by parafilm to prevent CO₂ leakage and it was placed into microscopy chamber tempered to 37 °C. NSC348884 was added just before the start of the measurement. Fluorescence images were processed by the FluoView software FV10-ASW 3.1.

Vero or Vero-d2EGFP cells were seeded in 500 µL volume to black-walled 24 well plates with glass bottom (Cellvis, Mountain View, CA) at a density of 100,000 cells per well. After an overnight incubation at 37 °C with 5% CO₂, the cells were incubated in serum-free Ham’s F-12 medium containing 20 μg/mL of cycloheximide (Sigma Aldrich, St. Louis, MO) or the stated toxin dilutions. Following incubation, the cells were washed with phosphate-buffered saline (PBS) and then bathed in PBS for EGFP measurement using a Synergy H1 Multi-Mode Microplate Reader (Biotek, Winooski, VT) with bottom optics position and 485 nm excitation / 528 nm emission filter set. For subsequent cytofluorometry analysis, the cells were detached using PBS without calcium and magnesium (GE Healthcare, Logan, UT). EGFP fluorescence was measured using an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA). All experiments recorded 10,000 events. For quantification of both plate reader and cytofluorometry data, background levels of autofluorescence from the parental Vero cells were subtracted from the experimental measurements. Background-subtracted data from treated samples were expressed as percentages of the control value obtained from untreated Vero-d2EGFP cells.

U2OS cells stably expressing GFP-ATR were seeded into 24-well plates with a glass-bottom (Cellvis) 24 h before laser micro-irradiation in a density of 6 × 105 cells/mL. After seeding the cells into the 24 well plates, the specimen was first placed on an equilibrated bench for 20 min at room temperature (RT) to ensure equal cell distribution and then placed into an incubator. CuET was added to cells 5 h before micro-irradiation in final concentrations of 250 nM and 500 nM. Twenty minutes before laser micro-irradiation, cells were pre-sensitized towards UV-A wavelength by 20 µM 8-Methoxypsoralen (8-MOP) and placed inside Zeiss Axioimager Z.1 inverted microscope combined with the LSM 780 confocal module. Laser micro-irradiation was performed at 37 °C via X 40 water immersion objective (Zeiss C-Apo 403/1.2WDICIII), using a 355 nm 65 mW laser set on 100% power to induce the DNA damage. The total laser dose that can be further manipulated by the number of irradiation cycles was empirically set to two irradiation cycles. Subsequent immunofluorescence detection and quantitative analysis of the striation pattern in photo-manipulated samples were essentially performed as described previously [21 (link)].