Cells labeled with LysoTracker (Invitrogen, L7528) and MitoTracker (Cell Signaling Technology, 8778) were transferred to a microscope stage and maintained at 37°C. Pseudo-TIRF microscopy (oblique illumination) experiments were performed using a X100 1.45 numerical aperture TIRF objective (Nikon) on a Nikon TE2000U microscope custom-modified with a TIRF illumination module as described [11 (link)]. Images were acquired on a 14-bit cooled charge-coupled device camera (Hamamatsu) controlled through NIS-Elements software. For live cell imaging, the images were recorded using 200–500-ms exposures depending on the fluorescence intensity of the sample. The track speed mean was then analyzed using Imaris as described before [11 (link),39 (link)]. Wild-type and ctns-/- cells were analyzed comparatively by maintaining exposure time and gain throughout the experiment. Length of mitochondria were analyzed using the “Measure” tool in ImageJ software.
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Lysosome and Mitochondria Imaging in Cells
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Corresponding Organization : Scripps Research Institute
Other organizations : University of California, San Diego
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Variable analysis
independent variables
- Wild-type and ctns-/- cells
- Track speed mean
- Length of mitochondria
- Exposure time
- Wild-type cells as a positive control
- Ctns-/- cells as a negative control
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