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…
Disclosures
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 |
Cite This Article
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).