Acridine Orange base 

Acridine Orange base is a cell-permeable fluorescent dye that stains organisms (bacteria, parasites, viruses, etc.) bright orange and, when used under appropriate conditions (pH=3.5, Ex=460 nm), distinguishes human cells in green for detection by fluorescence microscopy. Acridine Orange base fluoresces green when bound to dsDNA (Ex=488, Em=520-524) and red when bound to ssDNA (Ex=457, Em=630-644) or ssRNA (Ex=457, Em=630-644), also can be used in cell cycle assays^[1][2][3].

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Differential staining of DNA and RNA of unfixed cells^[1]:
(1) Set up the flow cytometer with excitation at 488 nm, using emission filters and a dichroic mirror that discriminate green fluorescence (measured at 515-545 nm) and red luminescence (measured preferably above 640 or 650 nm).
(2) Transfer a 0.2-mL aliquot of the original cell suspension to a small glass or plastic tube (e.g., 2- or 5-mL volume). Chill on ice.
(3) Gently add 0.4 mL ice-cold cell permeabilizing solution. Wait 15 s, keeping cells on ice.
(4) Gently add 1.2 mL ice-cold Acridine Orange base staining solution. Keep cells on ice in the dark.
(5) Measure and record cell fluorescence in the flow cytometer during the 2 to 10 min after addition of Acridine Orange base staining solution.
2. Differential staining of fixed cells^[1]:
(1a) For cells in suspension culture or hematologic samples: Rinse cells once with ice-cold PBS and suspend in ice-cold PBS at about 10⁶ cells/mL.
(1b) For cells attached to tissue culture plates: Collect cells from flasks or petri plates by trypsinization, pool the trypsinized cells with cells floating in the medium (mostly detached mitotic and dead cells), and rinse once with medium containing serum to inactivate the trypsin. Suspend cells in ice-cold PBS at about 10⁶ cells/mL.
(1c) For cells isolated from solid tumors: Rinse cells free of any enzyme used for cell dissociation and suspend in ice-cold PBS at about 10⁶ cells/mL.
(2) With a Pasteur pipet transfer 1 mL cell suspension to a 15-mL conical glass tube containing 10 mL ice-cold 70% ethanol. Fix cells ≥2 h on ice.
(3) Centrifuge tubes 5 min at 300 × g, 4 °C. Remove all ethanol, rinse cells once with ice-cold PBS, and suspend in ice-cold PBS at a density of < 2 × 10⁶ cells/mL.
(4) Withdraw 0.2 mL cell suspension (≤ 2 × 10⁵ cells) and transfer to a small tube (e.g., 2 or 5 mL volume). Chill on ice.
(5) Add 0.4 mL ice-cold permeabilizing solution. Wait 15 s, keeping cells on ice.
(6) Add 1.2 mL ice-cold Acridine Orange base staining solution. Keep cells on ice.
(7) Measure and record cell fluorescence in the flow cytometer during the 2 to 10 min after addition of Acridine Orange base staining solution.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

265.36

C₁₇H₁₉N₃

494-38-2

Solid

Yellow to orange

520/570

488

CN(C)C1=CC=C2C(N=C3C=C(N(C)C)C=CC3=C2)=C1

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Darzynkiewicz Z, et al. Differential staining of DNA and RNA. Curr Protoc Cytom. 2004 Nov;Chapter 7:Unit 7.3.  [Content Brief]

  [2]. Mirrett S. Acridine orange stain. Infect Control. 1982 May-Jun;3(3):250-2.  [Content Brief]

  [3]. Yektaeian N, et al. Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells. Heliyon. 2019 Dec 18;5(12):e03073.  [Content Brief]