We have been detecting LC3 lipidation and p62 reduction by western blotting in our studies. However, neither of these features are specific to macroautophagy. These changes do not always accompany double membrane-autophagosome formation (electron microscopy). Are there any other more specific biochemical assays being used?
Use GFP-LC3 plasmid and study puncta formation as upon induction of auophagy, diffuse pattern of LC3 changes into puncta. Study co-localization of LC-3 or p62 with lysosomes will be helpful.
I agree with Mohammad that LC3-GFP plasmid is a good way to do but again no method is the best one for detecting autophagy. EM is powerful but only limited area can be seen per view and you need to have extensive number of image to be sure about the change. The coming soon guideline from professor Klionsky can give you a better suggestion and I do hope it will be available next month.
Dear Afshin
My experience in past few years proved that macroautophagy detection is not as easy as many people think about it. We usually follow the following step by step procedure to confirm we have a real macroautophagy:
1- The first step is started with a routine WB analysis approval increase the ration of LC3 II/LC3 I, increase of Atg5-12 conjugation, and p62 status.
2- In the nest step we will carefully confirm we have a really autophagy Flux to make sure the finding in part 1 is not due to changes in biosynthesis of lipidated LC3. For this propose we will usually use Bafilomycin A1. Please be careful, Baf-A1 concentration should be set in each system and more importantly Baf-A1 is now considered a compound with several off-target effect so please be careful to set up a very careful dose evaluation for the system.
3- Next step is investigation of co-localization of LC3 puncta with lysosomes using lysotracker and LC3 or LC3 and LAMP-I. We will usually use confocal microscopy for that. Please also use Baf-A1 in this aspect to have a more stronger confirmation. We usually count LC3 puncta in 10 different field to have a better evaluation of the severity of macroautophagy.
4- TEM is strongly recommended. Please select at list 10 cells. Take different magnification and look at double membrane structure and also evidence of activation of lysosomes and localizations of lysosomes and phagosomes. In the 10 cells you selected count the event and make an average and compare with control. Please be careful for mis interpretation of the double membrane structures.
5- Please be advice that UPR/Autophagy is tightly linked and you can't direct them.
Good Luck
Saeid
Dear Dr. Samali,
I agree with said employing different markers and techniques to monitor various phases of autophagy. Our paper has been accepted recently with the details of monitoring autophagy robustly using flow cytometry and Live cell imaging. I shall upload it as soon as the galleys of the manuscipt is completed. I am providing you with the DOI of the paper and you may download it as soon as It is available. 10.1016/j.bbamcr.2014.12.022
Best,
jagan
Dear all, thank you so much for your replies. We have done imaging for LC3 puncta in the past. This technique also has the same shortcomings as western blotting for LC3 lipidation. I agree that there is no single best method for the detection of autophagy and as such multiple assays and methodologies should be utilized.
Jahan, I look forward to reading your paper.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques