Technical Reference #1828

1828.

Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells Maïte´ Courel; Alex Soler-Jover; Juan L. Rodriguez-Flores; Sushil K. Mahata; Salah Elias; Maïte´ Montero-Hadjadje; Youssef Anouar; Richard J. Giuly; Daniel T. O’Connor; and Laurent Taupenot, University of California San Diego, Journal of Biological Chemistry, 285(1828), (2010)
Link To Paper

Keywords:
Cell/Exocytosis; Cell/pH; Cell/Secretion; Methods/Fluorescence; Protein/Chimeras; Protein/Intracellular Trafficking; Subcellular Organelles/Vesicles; Chromaffin

Materials & Methods:
Total Internal Reflection Fluorescence Microscopy (TIRFM)— Cells expressing SgII-GFP were grown onto poly-L-lysine- and collagen-coated 35-mm glass bottom culture dishes (MatTek) and washed with serum-free DMEM prior to imaging performed at room temperature on a custom system including an inverted Nikon TE300 fitted with a 60 high numerical aperture objective lens (Nikon Plan Apo N.A 1.45). Excitation was produced by an argon/krypton ion laser (Melles Griott Multiline) and incident light for total internal reflection illumination was introduced from the high numerical aperture objective lenses. Images were acquired on an EMCCD Cascade II camera (Photometrics) controlled by Image-Pro Plus 5.0 software (Media Cybernetics). The value of the penetration depth of the evanescent wave used to excite GFP was estimated to be 140 nm in the z axis after calibration using 2- m fluorescent polymer microspheres. Exposure times were 100–200 ms and frames were acquired between 5 and 8 Hz in stacks of 200–600 images.

Microscopic Technique
TIRFM

Cell Type(s)
A35C-S7 clonal cell line