Glass bottomed tissue culture plates

Wide-field fluorescence images were obtained using the Cell^R system based on an Olympus IX81 fully motorized inverted microscope (60X PlanApo objective, 1.42 NA) fitted with an Orca-AG CCD camera (Hamamatsu) driven by the Cell^R software. Live-cell imaging was carried out using the Cell^R system with rapid wavelength switching. For time-lapse imaging, cells were plated on glass-bottomed tissue culture plates (MatTek, Ashland, MA) in medium containing 10% FBS at 37°C. The microscope is equipped with an incubator that includes temperature and CO₂ control (Life Imaging Services, Reinach, Switzerland).
For counting of nuclear speckles, SRSF7-GFP cells were untransfected or transfected with either RFP-CLK1 or RFP-TNPO3. 24 hrs post-transfection, cells were fixed and imaged under the same conditions. Nuclear speckles detection was performed using the ImageJ "find maxima" function, with noise tolerance value set to 200. For measuring the nuclear to cytoplasmic (n/c) ratio of SRSF4, the average fluorescence intensity of GFP-SRSF4 or GFP-SRSF4 with no RS domain was measured in a representative area in the nucleus and cytoplasm of the cells using the ROI measurement function in Xcellence software.

Wide-field fluorescence images were obtained using the CellSens system based on an Olympus IX83 fully motorized inverted microscope (60× UPlanXApo objective, 1.42 NA) fitted with Prime BSI sCMOS (Teledyne) driven by the CellSens software. Colocalization analysis of two channels was performed using an ImageJ macro (Shav-Tal lab, Ramat Gan, Israel). SG area was analyzed using ImageJ. Live-cell imaging was carried out using the CellSens system with rapid wavelength switching. Cells were plated on glass-bottomed tissue culture plates (MatTek, Ashland, MA) in medium containing 10% fetal bovine serum. Imaging was carried out at 37°C, using an incubator that includes temperature and CO₂ control (Life Imaging Services, Reinach, Switzerland). Live-cell movies were edited by ImageJ.

Wide-field fluorescence images were obtained using the Cell^R system based on an Olympus IX81 fully motorized inverted microscope (60X PlanApo objective, 1.42 NA) fitted with an Orca-AG CCD camera (Hamamatsu) driven by the Cell^R software. Live-cell imaging was carried out using the Cell^R system with rapid wavelength switching. For time-lapse imaging, cells were plated on glass-bottomed tissue culture plates (MatTek, Ashland, MA, USA) in medium containing 10% FBS at 37 °C. The microscope is equipped with an incubator that includes temperature and CO₂ control (Life Imaging Services, Reinach, Switzerland). For long-term imaging, several cell positions were chosen and recorded by a motorized stage (Scan IM, Märzhäuser, Wetzlar-Steindorf, Germany). Mitosis movies were recorded overnight by capturing images at selected areas every 30 min. Some of the mitosis movies were acquired using an Olympus IX81 microscope (636 Plan-Apo, 1.4 NA) equipped with an EM-CCD (Quant-EM, Roper) and an XY&Z stages (Prior), driven by MetaMorph (Molecular Devices). Experiments were performed at 37 °C with 5% CO₂ using a live-cell chamber system (Tokai).