Acridine Orange intercalates into nicked or damaged DNA and will foluresce "Red" on 488 blue laser.  Undamaged DNA or sperm cells with correctly packaged DNA will resist intercalation and the compound flouresces "green".  In this way the ratio of red to green can be calculated as the DNA fragmentation index (DFI).  I use AO from sigma aldrich (SIAL) or bioscience also have it. Important to use very high purity and concentration of stock solution should be 1mg/ml.  maintain AO powder at -20.

Hello, Javed

than you very much these are very useful for me

From experience, AO didn't work too well in ram and stallion spermatozoa. Also, if you're hoping to use FACS be warned that the AO stain seems to leave behind fluorescence in the machine for weeks after you have ran the sample through it. I've used TUNEL - more expensive but much more specific and the controls always worked as expected. I also found that the TUNEL stain from Roche could be diluted down so that you can use a dilution rate of 1 in 2 for the enzyme. Good Luck!

Hello Tarun

You may wash and prepare a final concentration of spermatozoa 30x106 cells/ml. You fix X 4 hours smears of spermatozoa with Carnoy’s solution (3 methanol: 1 crystalloid acetic acid) which must be prepared the day of the experiment (Tejada et al. 1984). After fixation, you stain the smears of spermatozoa with a solution of acridine orange (A6014) and evaluate under fluorescence microscope. You have to assess at least two hundred spermatozoa per slide in ten different optical areas for determination of percentage of spermatozoa with denaturated DNA.

Good luck

Thank you Clarie your suggestion will be helpful to me in near future. For now i will use fluorescent microscope.

Thank you Maria, for your reply and i will do the same and come to you if any difficulty.

Hi Tarun. Can you read Spanish. I can send you some papers or the summaries in English. Good luck.

hi tarun

i done AO staining in rat semen. acridine orange is used to stain sperms.

fluroscent-CX41- Olympus microscope is neccesary for the evaluation of abnomal headed sperms.

Hello Sir Humberto,

Send papers i will go through it

hello Madam,

Kanchana Ganga, Can you send detail procedure and exact formula of stain

As soon I ´ll go back to the lab I send them, we are now on vacations. 

thank you sir

Acridine Orange intercalates into nicked or damaged DNA and will foluresce "Red" on 488 blue laser.  Undamaged DNA or sperm cells with correctly packaged DNA will resist intercalation and the compound flouresces "green".  In this way the ratio of red to green can be calculated as the DNA fragmentation index (DFI).  I use AO from sigma aldrich (SIAL) or bioscience also have it. Important to use very high purity and concentration of stock solution should be 1mg/ml.  maintain AO powder at -20.

I stained rat sperm for acridine orange. I can see even the tails are stained red. I do not think it is right. What could be the reason?