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

Published: April 30, 2023

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…

Divulgazioni

The authors have nothing to disclose.

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

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Citazione di questo articolo
ADI-based Autophagy Induction in Prostate Cancer Cells: An Enzyme-based Technique to Measure Autophagic Response in Cells. J. Vis. Exp. (Pending Publication), e20402, doi: (2023).

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