We have performed AO assay on human ejaculated sperm. Everything seems, that works well, however we observed und microscope every cells stainned green and moreover there are two distinct population of sperm. 1) bright green fluorescence in acrosomal cap and 2) bright green fluorescence in post-acrosomal region.
Does anybody else has observed this? Please, could anybody give me suggestion what could be reason of this patterns?
Thank you.
Ondřej
Dear Ondrej,
prior to answer your question, please give me following infos:
- Are you using AO als base or AO hydrochloride (e.g. commercial sample)?
- Do you perform your test as vital staining (in vivo) or in vitro after fixation?
- What a concentration are you using?
- Pleae give me infos about your buffer (e.g. 7,2 or 7,7) and your cell medium.
- Please search the pKb value of AO , HCl in the web. it is important for your staining.
-Please open the following link and read the papers in it.
https://scholar.google.de/scholar?hl=de&as_sdt=0,5&q=sperma+AO+staining
good luck
JRG
Dear Mr. Grezes,
thank you kindly for your answer. I am responding now, because my colleague who actually do this assay have been finding information which you asked.
There is answer (however without dissociation constants we have to search for it):
AO – Acridine orange hemi (zinc chloride) salt (Sigma cat. no.158550)
Fixation - Carnoy‘ s fix minimum 2 hours (usually overnight)_+4°C
Concentration AO : 0,1 mg / ml in buffer pH 6 (0,2M NaH2PO4, 1mM EDTA, 0,15M NaCl, 0,1M citric acid)
Washing Cell buffer: PBS pH 7,4 (150mM NaCl, 17,6mM NaH2PO4* 2H2O)
Our team express you big thanks for you suggestions.
My best regards,
Ondrej
Dear Ondrej,
Acridine orange is a nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic attractions respectively. When bound to DNA it shows an emission maximum at 525 nm (green fluorescence). When it associates with RNA, the emission maximum shifts to 650 nm (red fluorescence). This is due to the electrostatic interactions occurring when the acridine molecule intercalates between the nucleic acid base pairs.
Acridine orange can also accumulate in acidic compartments such as lysosomes or organelles derived from them, like acrosomes. In these low pH conditions, the dye will emit orange light when excited by blue light.. It can be also used to evaluate the integrity of sperm chromatin (Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation, K.R. Chohan, J. T. Griffin, D. T. Carrell, Andrologia, 36, 5, 321-326, 2004). The result of acridine orange staining is significantly influenced by the method of fixation (Carnoy´s fluid, glutaraldehyde, cryopreservation.
Best regards,
Daniela
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