Coolled pe 1 excitation system

Cells grown on coverslips were fixed and permeabilized simultaneously in PTEMF buffer (0.2% Triton X-100, 20 mM PIPES, pH 6.8, 1 mM MgCl2, 10 mM EGTA and 4% formaldehyde). Cells were stained with mouse anti-myc 9E10 monoclonal antibody (culture supernatant, 1:2) and human CREST autoimmune serum (1:2,000; Immunovision). DNA was visualized with 4′,6-diamidino-2-phenylindole (2 μg ml⁻¹). All primary antibodies were detected with Cy2/Cy3-conjugated donkey antibodies (Dianova). A Deltavision microscope (Applied Precision) was used for immunofluorescence processing and image acquisition60 (link). For time-lapse microscopy, all treatments within a single experiment were performed simultaneously. Cells were imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 excitation system and a 20 × /0.75 air Plan Apo objective (Nikon). During imaging, the atmosphere was maintained at a temperature of 37 °C, humidity 60 and 5% CO₂. Images were captured at 5-min intervals for 22 h at multiple positions. Green fluorescent protein and mCherry fluorescence images were acquired at each time point with 30 ms and 60 ms exposure times, respectively. mCherry fluorescence was imaged only every five time point to monitor transfected cells. MetaMorph 7.7 software (MDS Analytical Technologies) was used to collect and process data.

hTERT-RPE1 cells stably expressing histone 2B-GFP were seeded in six-well chambers, treated as indicated and imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 excitation system and a ×20/0.75 air Plan Apo objective (Nikon). Images were acquired at multiple positions every 20 min. To collect and process data, ImageJ 1.44o software (Wayne Rasband, National Institutes of Health, USA) was used, respectively.