ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells

In dit artikel

Samenvatting

Source: Changou, C. A. et al. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy. J. Vis. Exp. (2013)

In this video, we demonstrate arginine deiminase-based autophagy induction in prostate cancer cells. Further, advanced fluorescence microscopy can be used to visualize the changes associated with autophagy induction, including the formation and distribution of autophagosomes and lysosomes and their fusion into autolysosomes.

Protocol

1. Preparing Cells for Live Imaging

Grow CWR22Rv1 cells expressing green fluorescent protein-coupled Light Chain 3 (LC3-GFP) in RPMI culture media containing 10% FBS and 1% antibiotics on 35 mm poly-D-lysine coated glass-bottom culture dishes. Cells should be plated at sufficient density to facilitate rapid proliferation, but not so much that cells are overgrown and clumped by the time of imaging.
Treat selected cell samples with ADI (0.3 μg/mL) in PBS.
Approximately 1 hr prior to imaging, dilute 1.5 μl of LysoTracker Red DND-99 (Invitrogen, CA) with 20 mL RPMI containing 10% FBS and 1% antibiotics. U....

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Resultaten

Fluorescence microscopy of cell structures, highlighting protein localization and cellular interactions.
Figure 1. Autophagosome and lysosome distribution in the cell. A. Top) Side-by-side comparison of images acquired and reconstructed in DV mode (simulated widefield deconvolution, left) and structured-illumination mo.......