I have tried to use nonyl acridine orange stain for my studies. I would suggest you to seed the required number of cells for staining. Give the treatments as per your protocol. On the day of staining, remove all media, wash the wells with cold PBS and discard. use fixative now. then wash with PBS, stain the cells for 15 mins inside incubator. then remove the stain, wash with PBS and cover with cover slip. observe under microscope. hope this will help.

This dye is usually used as a mitochodrial probe (not for autophagy)

Acridine orange is used in autophagy assays. It crosses into lysosomes (and other acidic compartments) and becomes protonated. The protonated dye stacks and stacked acridine orange emits in the red range. Acridine orange not in an acidic compartment will emit as green. But keep in mind that acidotropic dyes like AO only stain late autophagic vacuoles.

Any suggestion about concentration of this dye?

be careful with AO because easily bind to DNA specially if you incubate dead cells

Pietro Di Fazio thank you very much for an advice

Your welcome

Autophagy is characterized by increased formation of AVOs (lysosomes and autophagolysosomes). AVOs can be quantified by flow cytometry after staining the cells with AO (acridine orange). AO is a weak base that accumulates in acidic spaces and fluoresces bright red. The AVOs can be quantified based on the fact that the increase in intensity of the red fluorescence is proportional to the degree of acidity. For flow cytometry you can use 1 µg/ml, for 15 min at 37°C. Please remember that this is an old method, and can be used only as a supplementary result to show autophagy.

I would recommend using Acridine orange for detection of autophagy. This dye becomes proton trapped within lysosomes and when imaging lysosomes you will induce a photo-damaging reaction of the lysosomal membrane (it works like a photo-sensitizer). It's better to use more specific procedures like GFP-tagged LC3/p62 for live imaging or antibodies against these proteins in immuno stainings.

Best

Jesper

Hi, you can use Dansylcadaverine (MDC). The autofluorescent marker that preferentally accumulates in autophagic vacuoles. Go for the microscopic study of the cells after probing with MDC solution. MDC accumulation in auotphagic vacuoles is due to ion trapping as well as due to specific interactions with vacuole membrane lipids. Furthermore why not trying immuno-fluorescence with anti LC3 ab !! . It will detect the autophagosome-lysosome fusion

Sorry I meant:

I would not recommend using Acridine orange for detection of autophagy. This dye becomes proton trapped within lysosomes and when imaging lysosomes you will induce a photo-damaging reaction of the lysosomal membrane (it works like a photo-sensitizer). It's better to use more specific procedures like GFP-tagged LC3/p62 for live imaging or antibodies against these proteins in immuno stainings.

Best

Jesper

Jesper Nylandsted ;-), thaks for your advice, but I just search a simple and really cheap method to confirm autophagy using fluorescent microscopy.

Vinoth K.M. Khandelwal: Have you ever used AO in fluoroscent microscopy?

Atish Ray: Nice alternative. I will consider that

Jakub Nowak : Hope you will get better result . (MDC is available in powder form manufacturers such as Sigma) . Thanks

Sorry Jakub, I didnt use AO for microscopy. But, I guess it should work, if you try with the similar concentration (may be for 15-30 min incubation) for live cell imaging. If you try to do, then please let me know what you observed.

Sure

In your interpretation don't forget that acridine orange is also used to stain dying cells. White et al. 1994 Brodsky 2004

You can use AO for microscopy, I incubated cells for 30 minutes. But the signal quenches very fast, and it is definitely not a marker for autophagy. IF for LC3 would be better.

Mohammad Ishaq: I have already done elecotron microscopy and I supposed that autophagy may be occure Have you any photos of autophagosome staining using AO?

Sorry, I´m late on that topic, but why using this dye and not protein marker as LC3, P62...

Casadei Nicolas: I found in my lab AO and I was wondering if I could use it for autophagy detection. These proteins markers I don't have in my lab

That make sens.

From my point of view, that could provide a fast estimation if autophagy could be disregulated.

Like Nadia or Mohammad, I would confirm these results using protein reporter.

Good luck and best regards!

Don´t forget to share the results!!!

This article gives you reference for both AO experiment and electron microscopy. Remember that AO detects all acidic vesicular organells (AVO), which includes lysosomes, late autophagosomes and late endosomes. "Oxidative stress induces autophagic cell death

independent of apoptosis in transformed and cancer cells http://www.ncbi.nlm.nih.gov/pubmed/17917680 "

And this article is a good one (important) to know more about the methods for evaluation of autophagy "Guidelines for the use and interpretation of assays for monitoring

autophagy in higher eukaryotes http://www.ncbi.nlm.nih.gov/pubmed/18188003 "

Vinoth thank you very much These publications should help me

Check this articles: http://www.ncbi.nlm.nih.gov/pubmed/20144757

http://www.ncbi.nlm.nih.gov/pubmed/20225337

Tommorow i will send you the protocol which we used, but like it was previously written AO can be only use for check the accumulation of acidic vesicles. It can depend on block in some later steps of autophagy process. But if u see vesicles in electron microscopy and if you will confirm it by AO, i think its resonable buy some LC3/p62 dependent assay. Tell, what exactly you do?

Here you are some photos which we take:

http://hit4you.pl/?di=R11M (autophagy positive)

http://hit4you.pl/?di=FZNR (nagative control)

Adam Thank you very much

Adam I suggested your protocol to my collegue and she try tu use this procedure. The results are promising. It's a good procedure for confirmation of autophagy that we previously found using TEM

Probably soon we will publish these results and if u are interested i will send u our publication

It will be a pleasure to see yours results

First results ;-) Thanks to Adam

Photo

Nice staining!

And how is looking a negative control ??? :)

Negative control of staining procedure ?? or you thing about control to my experiment (control cells = untreated cells) ??

I mean control, sorry for the typing error (I edit the past post)

I was asking how you plan to prove that what you observe is really specific?

To illustrate what I mean, it could be possible that the dye does not enter cell membrane. Then the dye accumulate on cell membrane at specific location and make these "dots". So a good negative control would be have cell without autophagic vacuole.

Best

What type of cell line is this one?

Adam: It is CHO AA8 cell line

Nicolas: I do not have a negative control of cells without autophaigic vacuole. Even in my control cell some acridine signal was abserved By the way this procedure was performed just to confirm observations in TEM. My controls also have some autophagic structures. But we observed changes after treatment procedure. When our articles will be accepted i will send you a proof if you are interested.

I'm setting up AO staining to detect autophagy. The first observation, Tamoxifen-induced autophagy on MCF-7 cells had many huge cells (figure attached). Is that normal in autophagic cells or not?

Dear Hoang,

you should open a new post because your question is off topic.

I contact the webmaster, your question should be redirected automatically soon.

Thank you Casadei Nicolas

I am concerned about the fact that lysosomes after AO staining also appear red under 488nm excitation. How would you differentiate the autophagic vacuoles and lysosomes?

The good negative control can be chloroquine treated cells.

Dear all!!

I have done acridine orange staining to visualise autophagy in THP-1 cells. I used FITC channel in fluorescence microscopy. I see the green spots in the cells in rapamycin treated cells. How to get the orange spots?? What wavelength i must use for microscopy??

Alok, to observe the orange/red signal, you should use the 488nm excitation/615nm emmission, preferably a long-pass filter.

Thanks Siyuan!!

Hey Jakub,

I am also doing AO staining in prostate cancer cells to see autophagy. Can you please share your protocol to me? I would be happy to use your protocol. Thanks!

AO cannot detect autophagosome or lysosome in fixed cell.

Live cell stained should be better to detect red-orange signal.

Dear sir

With Ethanol seem to suitable

Best wishes

Dear Jakub,

as I know Acridine orange is used for lysosomes detection.you can use CYTO ID (Enzo life sciences ) for specially  autophagy detection 

thanks

Actually acridine orange is useful for detection of lysosome, simultaneous experiment is needed with MDC (Mono Dansyl cadaverin ) and microscopy is recommended along with LC3b IF staining. Taking together story of different stages of  autophagy(i.e  autophagosome maturation, fusion  with lysozome etc)  will be convincing. (Instead Acridin orange, Lysotracker dye can also be used to detect lysozome)    

Dear Friends,

Please read this article to get updated information on Acridine orange staining for autophagy assays.

http://jcs.biologists.org/content/joces/129/24/4622.full.pdf?with-ds=yes

Regards,

Puru

The paper added by Puru is really interesting but I would be really careful! IT is an indirect measurement based on the lysosome accumulation...a direct measurement of autophagosomes would be the best option.

I agree with Pietro. Acridine orange method is just for preliminary studies.

.

I have seen the expression of increased autophagy related proteins, however examining the AVO by AO I flow cytometry I found a decrease in red fluorescence after 48 hr treatment of compound.

I wonder that whether AO is a good indicator for autophagy.

I am doing AO staining in colon cancer cells to see autophagy. Can you please share your protocol to me? Thank you.

Aftab Nadeem

For autophagy study, mRFP-GFP-LC3 and GFP-LC3 labeling system 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.

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

I did that, the experiement need some fines seting in flow cytoetry but that works very well and the level of cell death could be determine at the same time. Please see Rainey et al., SCIENTIFIC REPORTS 2016, 7, 4028. You can contact me by [email protected] for more details.