Hi Li He Chloroquine as an Autophagy inhibitor functions via blocking the fusion of the Autophagosome with the Lysosome by increasing the intralysosomal pH. This event leads to the accumulation of LC3 inside the Autophagosome which can be detected via western blot. However, it is important to perform this experiment under controlled condition otherwise, the interpretation of LC3 level becomes complicated.

However, the most accurate way to measure autophagy is via the autophagic flux assay. This can be defined as the formation of new double-membrane vesicle-like structures called "autophagosomes" and their subsequent fusion with the lysosome for degradation of the autophagosomal contents.

To make this clearer, here is a great article written by Dr Christina Towers for Novus Biologicals about Measuring Autophagic Flux with LC3 protein levels: The do's and don'ts. Available online:

https://www.novusbio.com/antibody-news/measuring-autophagic-flux-with-lc3-protein-levels-the-dos-and-donts

I am sure you will get things clearer after reading this article. Best of luck.

@ Lukman Afolabi your answer does help me a lot, thanks very much. And besides the advise(last 2 hours to add CQ) of Dr Christina Towers , there are also some researchers recommending pretreating. Any difference between the two methods?

I will suggest you add the drug towards the last few hours depending on the time course you are considering. There are always varying opinions but I think this method should be fine for your experiment.

@ Lukman Afolabi Too much appreciation. I will try your recommendation.