Acridine orange - NAO vs acridine orange (Nov/14/2009 )

I want to check if I have autophagy in drug-treated cells. I have nonyl acridine orange (NAO) available for the determination of mitochondrial. Recently I found that acridine orange can be used to detect autophagic vacuoles. Is there any difference between NAO and the acridine orange used for autopgahy detection? Or can I use NAO?

And NAO is the same that 10-Nonylacridine orange bromide?

And NAO is the same that 10-Nonylacridine orange bromide?

Yes it is. 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.

Thanks.

So I won't be able to detect autophagy vacuoles with NAO, right?

Unfortunately not.

miBunny on Nov 17 2009, 04:30 AM said:

Do you have idea on what concentration of AO is good for detecting autophagy in cells using FACS. In literature there is from 100 ng/ml to 100 mcg/ml, incubated for 15 min. Also, is it required to use compensation for detecting green and red signal from AOl? Because there is signal in all channels, from green to extreme red.

Is it possible to detect the autophagy proteins using FACS? Example: detecting LC3B, Beclin, ATG 5 or 12 or 7, using the primary antibodies (used for western blot), conjugated with fluoresence labelled secondary ab?


Thanks