Computational protocol: Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

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Protocol publication

[…] Cells were imaged in pre-warmed HBSS media on a Nikon Ti-E inverted microscope with a 100×1.49 NA objective in an environmental chamber heated to 37°C equipped with an Andor iXon X3 EMCCD camera (Andor Technology, Belfast, UK). Proinsulin-eGFP, GFP-tubulin and EB3-GFP samples were imaged using total internal reflectance microscopy excited with a 488 nm solid state laser (Andor Technology, Belfast UK) and emission collected at 525 nm at 19 fps, 10 fps and 5 fps, respectively. Images were collected in basal, low glucose conditions (2.8 mM glucose) before stimulation with glucose to a final concentration of 20 mM. Cells were imaged between 3 and 20 minutes after glucose addition. Insulin granules’ motions were tracked using the Image J v1.43 plug-in, SpotTracker2D (National Institutes of Health), allowing us to track the granules position at sub-pixel resolution in every frame of the movie so that mean square displacement (MSD) analysis could be performed (see below). Out-of-focus granules and granules which could not be spatially resolved from one another were eliminated from analysis. EB3-GFP point-to-point velocities were determined using the ImageJ 1.43 plug-in, MTrackJ.Imaging and tracking of insulin granules in fixed cells were used as a control for stage drift in our imaging set-up and to determine our imaging resolution. To prepare these fixed cells, INS-1 cells were transfected with proinsulin-eGFP as described above. 24 hours after transfection cells were fixed with 4% paraformaldehyde in 1×PBS for 15 minutes at room temperature. Cells were washed three times in 1×PBS and stored at 4°C. Samples were first imaged by maximum projection through time to visually assess for the occurrence of stage drift. MSD analysis was subsequently performed and alpha values (diffusive exponent, see below) for fixed samples was determined to be 0.05, indicating negligible stage drift. To determine our tracking spatial resolution, granules were imaged and tracked as with live cells. Tracking resolution of 23 nm was defined as the standard deviation of the X and Y coordinates from stationary granules in fixed cells. […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Chemicals Glucose