Bafilomycin A1, chloroquine and 3-MA can inhibit autophagy at different stage. For autophagy study, you could try mRFP-GFP-LC3 and GFP-LC3 labeling system, which can detect the intensity of autophagy flux in real-time, in which GFP and/or RFP tags are fused at the N-termini of the autophagosome marker LC3. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not. Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.

You could find more on this website: www.genemedi.net/i/autophagy

Genemedi provides the production service of AAV, adenovirus and lentivirus encoding mRFP-GFP-LC3 or GFP-LC3, suitable for monitoring the intensity of autophagy flux in real-time in vivo or in vitro.

Attached file is an image of mRFP-GFP-LC3 labeled A549 cells with nutrition deprivation.

I use chloroquine in my research and don't have big problems.

In fact there is a number of different autophagy inhibtors. You can find them in this review (see attach).

3-methyladenine (3-MA) is commonly used as autophagy inhibitor, but it's important to know the mechanism by which it inhibits it.  3-MA is actually a PI3K inhibitor and it is inhibiting autophagy by blocking the formation of autophagosomes. 

As Stanislovas mentioned, chloroquine is another autophagy inhibitor, however the mechanism by which it inhibits autophagy is completely different and it mainly block late autophagy. Chloroquine accumulates inside the acidic parts of the cell (lysosomes) and raises their pH, that way it inhibits autophagosome-lysosome fusion and degradation. This leads to accumulation of autophagosomes in the cell.

 Another autophagy inhibitor that works in a similar  way as chloroquine is bafilomycin A1. Bafilomycin block the fusion between autophagosomes and lysosomes by inhibiting vacuolar H+ ATPase. 

Inhibitors of autophagosome and lysosome fusion are commonly used as tools to prove the increase of autophagic flux. 

In the guidelines attached by Stanislovas and Manish you can get more in depth explanation about all of that as well as more inhibitors suggestions.

Depending on the purpose of your experiment you can select the most suitable autophagy inhibitor. If I may suggest to you, it's highly recommended to confirm that autophagy inhibition is actually occurring (e.g. by western blotting or confocal) with/without starvation, in that way you can also select the best concentration of inhibitor and duration of incubation.

Keep in mind that all autophagy inhibitors are also affecting the cells in various other ways, that can change your expected results. 

There is a nice kit called Autophagy Watch (Code No.8486 from MBL) which includes already diluted Chloroquine and Bafilomycin (x1000) as well as antibodies for western and immunocytochemistry.

Good luck!

I prefer to use CQ (chloroquine) in all of my researches about autophagy inhibition. The using of CQ is quite easy, since it is easy dissolve in water (unlike 3-MA in DMSO). One time I have been used 3-MA, actually I did not like to work with it. Bafilomycin is another choice for you.

Regards and good luck!

The attached article could be of help.

Robert

Chloroquine  works well to inhibit autophagy

A good solution for inhibiting just the autophagy itself without inhibiting the kinases upstream is also using ammonium chloride that prevents lysosomal acidification.

Thank you all for such amazing very helpful answers.

The problem of using chloroquine or ammonium chloride is that both of them prevent the acidification  of the lysosomes not to interact directly with the autophagosome formation as 3-MA does.

Fortunately, I tried 3-MA and it worked fine, but it is really hard to handle it especially in big experiments as it solidifies very fast at room temperature and needs 60 C to be in a liquid form. 

Dear Eslam

I also had problems with 3-MA so I tried Wortmannin, and LY294002 which are also PI3K inhibitors and prevent autophagosome formation. However, they also dissolve in DMSO like 3-MA but so far I haven't got problems handling LY294002.

Bafilomycin A1 is a known inhibitor of the late phase of autophagy. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes. in our lab for autophagy study we used usually Bafilomycin and 3-MA. 3-MA is actually a PI3K inhibitor and it is inhibiting autophagy by blocking the formation of autophagosomes. 

I recommend dilution solution of 3-MA in PBS, not DMSO. 3-MA is dissoved to 50 mM in PBS. If solidified, heat in 50 ℃. then store RT. 

Thank you all for the insightful comments and recommendations. I am currently working on Autophagy as well and your suggestions has been very handy...

Bafilomycin A1, chloroquine and 3-MA can inhibit autophagy at different stage. For autophagy study, you could try mRFP-GFP-LC3 and GFP-LC3 labeling system, which can detect the intensity of autophagy flux in real-time, in which GFP and/or RFP tags are fused at the N-termini of the autophagosome marker LC3. These biosensors provide an enhanced dissection of the maturation of the autophagosome to the autolysosome, which capitalizes on the pH difference between the acidic autolysosome and the neutral autophagosome. The acid-sensitive GFP will be degraded in autolysosome whereas the acid-insensitive RFP will not. Therefore, the change from autophagosome to autolysosome can be visualized by imaging the specific loss of the GFP fluorescence, leaving only red fluorescence.

You could find more on this website: www.genemedi.net/i/autophagy

Genemedi provides the production service of AAV, adenovirus and lentivirus encoding mRFP-GFP-LC3 or GFP-LC3, suitable for monitoring the intensity of autophagy flux in real-time in vivo or in vitro.

Attached file is an image of mRFP-GFP-LC3 labeled A549 cells with nutrition deprivation.

Hi Ruiyue Shi,

When you used mRFP-GFP-LC3 labeling system, have you seen green spots as well?

Xiaoqin Wu Yes, I can see lots of green spots.