Acridine Orange (Synonyms: NSC 194350)

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare Acridine Orange staining solution
(1) Acridine Orange dye stock solution: Use DMSO to dissolve Acridine Orange into a 1mg/ml stock solution.
Note: It is recommended that unused stock solutions be aliquoted and stored in the dark at -4°C or -20°C (stored under nitrogen), and avoid repeated freezing and thawing.
(2) Preparation of Acridine Orange dye working solution: Dilute the stock solution with a suitable buffer (such as serum-free medium, HBSS or PBS) to prepare an Acridine Orange working solution with a concentration of 0.5-20 μg/ml.
Note: Please adjust the concentration of the working fluid according to the actual situation, and use it now.
2. DNA and RNA staining of cells:
(1) Collect the cell suspension, transfer about 0.2ml/portion of the original cell suspension into a small glass or plastic tube with a volume of 2/5 mL, and place it on ice to cool;
(2) Slowly add 0.4 mL of pre-cooled cell permeabilization solution, and place the cells on ice for about 15 seconds;
(3) Slowly add 1.2 mL of pre-cooled Acridine Orange working solution, and incubate on ice in the dark for 2-15 minutes;
(4) Use a flow cytometer to measure and record cell fluorescence, excite at 488 nm, and distinguish dsDNA at green fluorescence (measured at 515-545 nm) and red luminescence (preferably at For RNA (measured above 640 or 650nm), the excitation/emission wavelength of Acridine Orange after binding to ssDNA is 460/650nm.
3. Cell adhesion staining (operated on ice)
(1) Culture adherent cells on sterile coverslips;
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment;
(3) Add 100 μL dye working solution from one corner of the coverslip, shake gently to make the dye evenly cover all cells, and incubate on ice in the dark for 2-15 minutes;
(4) Aspirate and discard the dye working solution, use culture medium to wash the coverslip 2 to 3 times, and observe with a fluorescence microscope.
4. Microscopy detection: Use a confocal laser scanning microscope (such as FV3000, Olympus) to observe cells, or quantify the total fluorescence of Acridine Orange in each frame through MetaMorph. Acridine Orange exhibits excitation/emission spectra of 502/525, 460/650 and 475/590 nm when bound to dsDNA, bound to ssDNA or RNA, and under acidic conditions, respectively.

Precautions:
① If cells need to be fixed and stained, it is recommended to use 70% ethanol for fixation and fix on ice for ≥ 2 hours. After fixation, rinse 1-2 times with pre-cooled PBS to remove all ethanol, and then stain according to the above steps;
② Fluorescent dyes all have quenching problems. Please try to avoid light to slow down fluorescence quenching;
③ For your safety and health, please wear a lab coat and disposable gloves.