Csu x1 spinning disc head

Live cell imaging of H2b-mcherry expressing MEFs was performed on glass-bottom 6-well plates (MatTek, Ashland, MA) using a Nikon Ti-E inverted microscope attached to a CoolSNAP CCD camera (Photometrics). Fluorescence and differential interference contrast (DIC) images were acquired every 7 minutes, and images were analyzed using NIS elements software (Nikon) and ImageJ software (NIH). For confocal imaging, MEFs were grown on 35mm glass bottom plates (MatTek, Ashland, MA) and DIC images were acquired every 5 minutes with the Ultraview Vox spinning disc confocal system (Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning disc head, and EMCCD camera (Hamamatsu C9100-13), and coupled with a Nikon Ti-E microscope. Image analysis was performed with Volocity software (Perkin Elemer). All imaging was carried out in incubation chambers at 5% CO₂ and 37°C.

Live cell confocal microscopy was carried out using the Ultraview Vox spinning-disc confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disc head and electron-multiplying charge-coupled device camera (Hamamatsu C9100–13) coupled to a Nikon Ti-E microscope. Cells were cultured on 35mm glass-bottomed dishes (MatTek) and imaged in live-cell incubation chambers maintained at 37°C and 5% CO₂. For GFP-Galectin3 imaging experiments, GFP-Galectin3 expressing MCF10A cells were treated with 0.5mM LLOMe for one hour to induce lysosome damage and then washed twice with media and cultured in growth media. For lysosome size control experiments, LAMP1-GFP expressing MCF10A cells were treated with 50μM monensin for two hours to enlarge lysosomes and were then washed twice with growth media and cultured in growth media. Dishes were immediately taken to the microscope at the indicated times for image acquisition. Image acquisition and lysosome size analyses were carried out using Volocity software (PerkinElmer).