Computational protocol: BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

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Protocol publication

[…] Quantification of the area of overlap in the cell periphery between two fluorescent labels or a fluorescent label and pigmented melanosomes was performed using ImageJ (National Institutes of Health) on wide-field fluorescence images using a method similar to that previously used with OpenLab software (). In brief, images were cropped to contain a single cell with the nucleus and perinuclear area removed, and binary images of fluorescence were generated by subtracting the local background before thresholding using parameters of a rolling ball radius of 10 pixels with smoothing disabled. Binary images of pigment granules from BF imaging were generated by manual thresholding. The Image Calculator function was used to generate an image representing the area of overlap between channels by multiplying the binary images for each of the two channels. The areas of overlap and of total fluorescent labeling in structures larger than five pixels were quantified using the Analyze Particles function; the ratio of overlap pixels to total fluorescent pixels in the channel of interest gives the percentage of overlap. [...] Cells were seeded in Matrigel-coated 35-mm glass-bottom dishes and transiently transfected with indicated GFP- or mCh/mRFP-fusion proteins using Lipofectamine 2000. At indicated times after transfection, cells in riboflavin-free RPMI (US Biological) containing 10% FBS were imaged on a Axiovert 200 microscope (ZEISS) equipped with a 63X Plan-Apo objective lens (NA 1.4), an UltraVIEW ERS6 spinning-disk confocal scan head (PerkinElmer), an environmental chamber at 37°C, an Orca ER CCD camera (Hamamatsu Photonics) and Volocity (PerkinElmer) software for image acquisition. In some experiments, an ORCA-Flash4.0 sCMOS camera was used instead. For imaging of cells expressing GFP-VAMP7 relative to melanosomes imaged by BF microscopy (; and Fig. S2 i), we used an Olympus IX71 spinning-disk confocal microscope equipped with a 100× Plan-Apo objective (NA 1.4), LCI Chamlide stagetop incubation chamber at 37°C/5% CO2, an ImageEM EM-CCD camera (Hamamatsu Photonics), and MetaMorph (Molecular Devices) software for image acquisition. Image sequences were further analyzed using ImageJ. All images were captured at ∼1 frames per second (fps) except for Video 9, which was captured at ∼1.4 fps. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications SPIM, Microscopic phenotype analysis