Please help. I am planning to do a Western Blot LC3 turnover assay to study autophagy flux in an A549 lung cancer cell line. I have only got leupeptin protease inhibitor in the lab. Is it sufficient as the only inhibitor of basal LC3 degradation in an LC3 assay? I would be also grateful if someone could suggest time points to study LC3 turnover in A549 cells when using a PP242 mTOR inhibitor. It will be my first attempt to study autophagy so I would be grateful for even some basic advice.
Hello!
WB for LC3 can be a pain. I found the following to work nicely:
- induction of autophagy with hunger medium or salt solution, e..g. Hank's Balanced Salt Solution (HBSS) for 0, 1 and 3 hours.
- co-treatment of cells with 100 nM Bafilomycin A1 to prevent the degradation of LC3 II
(but this depends on the background level of autophagy and is cell line-dependent)
- lysis of cells with RIPA and protease inhibitors
- load 5-10 ug (or more, depending on results)
- PVDF membranes!!! better than nitrocellulose...
- LC3 antibody from NovusBiologicals (rabbit polyclonal, catalogue no. NB-100-2220) at 1:500 to 1:1000 dilution in PBS-T with non-fat milk powder (5%w/v)
- detection with 2ndary AB and your preferred system of detection.
I hope this helps, otherwise feel free to contact me
Best wishes
I used chloroquine, it is a very safe way to be sure whether there is increased autophagic flux or not. I agree, it is very cheap and easy to prepare. but be careful to have the fresh one in each experiment. On the other hand, if you induce autophagy and used CQ, it is possible that you see more cell death, for that you can check my publication : ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages. in Researchgate web site I also have the full text.
You can even find useful references in the end related to this topic.
Good luck and Best Regards
Hello,
use Chloroquine and Bafilomycin A1. For both will give u the clear idea about autophagic flux. But before using them it will be better to do Time Kinetics of autophagic induction. Select a time point where maximum or medium autophagy is induced. Then you may add CQ or Bafilomycin A1 2-4 hr before the time point u have chosed. if there is an further increase in the accumulation of LC3-II , it clearly suggests there is an increase in the autophagic flux.
Good Luck.........
Thank you for all the answers! Has anybody done LC3 turnover in A549? I am thinking what experimental time points would work fine in my experiment. I have found some publication presenting LCII accumulation after 24 or even 48 h but after treatment with weak "natural" inducers. I am going to use very strong selective mTOR inhibitor so probably it will be rather few hours - am I right?
As far as I know, the duration of the bafilomicyn A1 treatment should be short (1-2 hours). I would recommend to read "Guidelines for the use and interpretation of assays for monitoring autophagy" from Klionsky.
Hi!
I used Bafilomycin for 24 hours and it was fine! Admittedly, I didn't do a time-course analysis.
Positive control for strong short-term induction (1-3 hours): incubation with HBSS
Attached is an article with ideas on how to do good autophagy experiments. I hope this helps!
And here is the link to the above mentioned guidelines:
http://www.landesbioscience.com/journals/autophagy/article/19496/?nocache=1834989330
(sorry, just the link, not the article so that LANDES Biosciences does not sue my pants off; the attached article is open access anyway)
Good luck!
You can download the paper for free at
http://gbb.eldoc.ub.rug.nl/root/2012/AutophagyKlionsky/
I hope it helps!
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