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

Abstract

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. …

Representative Results

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 mode using the OMX microscope (right). Scale bar represents 5 μm. B. Bottom) Small-scale colocalization of aut…

Materials

Arginine Deiminase (ADI)	DesigneRx
HEPES	Sigma	H4034
Casein	Sigma	C5890
Paraformaldehyde	Fisher	4042
Saponin	Sigma	S4521
Alexa anti-mouse 555	Invitrogen	A21422
Alexa anti-rabbit 647	Invitrogen	A21244
LysoTracker Red DND-99	Invitrogen	L7528
anti-Lamp1	DSHB	H4A3
anti-Cadherin	Cell Signaling	#3195
SlowFade Gold	Invitrogen	S36936
35 mm poly-d-lysine coated glass bottom plate	MatTek	P35GC-1.5-1.4-C
No.1, 22 mm coverslip	Corning	#2865-22
Microscope slides	Globe Scientific	1324G