I used acridine orange staining to detect autophagy. First, the cells were grown in RPMI 10%FBS in confocal dishes, treated with compound with or without with wortmannin (0.5uM). Wortmannin was used as an autophagy inhibitor. After 2h, 1ug/ml acridine orange was added directly to the medium and incubated for 20min at 37'C. Then, the cells were washed with PBS (3%FBS) for 2 times. Finally, PBS (3%FBS) was added to cover the cells and observed the staining under a confocal microscopy. (P.S: that protocol was originally used for lysosomal stability assay)
And unexpectedly, I found that wortmannin alone increased the red fluorescence.
In papers,
- some used acridine orange with Ethidium bromide to detect apoptosis
- some used it to detect autophagy
- some used it for lysosomal stability
I wonders how they analyse the outcome specifically.
Does it depend on difference in protocol??
Or is acridine orange staining alone not sufficient to determine the outcome?
Hi i only know this:
the monomeric binding of acridine orange to cellular DNA resunlts in green fluorescence emmision.While the polymeric binding to lysosomal structure results on red fluo.emission.
During apoptosis the red spectra emission remains unchanged while the green spectra is fainted (diminishing by time). We know our dye is although a membrane penetrant despite is weakly basic for the membrane of the lysosomes and enters and gets charged inside them and it traps in there. (state of protonation). Acridine orange during preliminary stages of apoptosis does not cause any change on the lysosomes while being accumulated in there. This goes against the necrotic death where acridine specifically is lost(diminished) from lysosomal structure and thats why we use this dye. To make recognition and discriminate the apoptosis versus necrosis an assay.
In necrosis we have red signal lost due to leaky membrane organelle (lysosome) where this results in increase of green signal VERSUS apoptosis where green signal is lost due to DNA breakdown where results in red signal increase as it is intact in lysosome and is accumulated."
Hope this helps and some more experienced to add more infos'
Best of luck !
Kat.
Hi i only know this:
the monomeric binding of acridine orange to cellular DNA resunlts in green fluorescence emmision.While the polymeric binding to lysosomal structure results on red fluo.emission.
During apoptosis the red spectra emission remains unchanged while the green spectra is fainted (diminishing by time). We know our dye is although a membrane penetrant despite is weakly basic for the membrane of the lysosomes and enters and gets charged inside them and it traps in there. (state of protonation). Acridine orange during preliminary stages of apoptosis does not cause any change on the lysosomes while being accumulated in there. This goes against the necrotic death where acridine specifically is lost(diminished) from lysosomal structure and thats why we use this dye. To make recognition and discriminate the apoptosis versus necrosis an assay.
In necrosis we have red signal lost due to leaky membrane organelle (lysosome) where this results in increase of green signal VERSUS apoptosis where green signal is lost due to DNA breakdown where results in red signal increase as it is intact in lysosome and is accumulated."
Hope this helps and some more experienced to add more infos'
Best of luck !
Kat.
https://www.nature.com/articles/s41598-017-13904-0
https://jcs.biologists.org/content/129/24/4622
http://jcb.rupress.org/content/216/4/1163
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332266/
Hello, it already established that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries etc. Necrotic necroptotic cells are characterized as those completely lacking cellular RNA but having a round, bright nucleus. In contrast, apoptotic cells also lose cytoplasmic RNA, but in vivo these cells had unique acridine orange emissions: the nucleus contained either red or fragmented green emission. These features were never observed in necrotic cells. Thus, changes in acridine orange spectrum differed with respect to necrosis necroptosis versus apoptosis, underscoring the ability of acridine orange to reliably distinguish modes of injury.
Thank you Kat Katerina Kouzari and Karen Karen A. Darbinyan for your sharing. I got new insights from you.
But there is one point still unclear. Does acridine orange accumulate in lysosomes in apoptotic cells more than in normal cells? Or is red signal similar to normal cells while only the green signal is decreased?
And about autophagy,
I just realized one thing. it has been reported that autophagy process involves lysosome and formation of autolysosomes ( fusion of autophagosomes and lysosomes). And these are acidic. So, may be, in autophagic cells, acridine orange also enters into these autolysosomes and shown in increase in number of red spots compared to normal cells.
If so, it will be really difficult to differentiate whether it is apoptosis or autophagy under acridine orange staining if ao staining was used as a preliminary experiment. It is just my assumption though. Could you enlighten me if it is correct or not??
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