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An In Vitro Technique to Visualize Exocytosis of Secretory Granules in Mast Cells

This video demonstrates a technique to visualize exocytosis in mast cells. The cells are incubated with FITC-dextran — a fluorescent reporter — which is taken up by cells inside secretory granules (SGs), where the acidic pH quenches its fluorescence. Upon stimulation of granule exocytosis, the medium's higher pH induces reporter fluorescence inside the fused SGs, which is visualized using a fluorescence microscope.

1. Preparations

  1. Preparation of RBL (RBL-2H3 mast cell line) culture media
    1. Mix 500 mL of low glucose Dulbecco's modified Eagle's medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.
    2. Filter the media by using a top-va.......

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Source: Klein, O., et al. Imaging FITC-dextran as a Reporter for Regulated Exocytosis J. Vis. Exp. (2018).

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