Acridine Orange base is a cell-permeablefluorescent dyethat 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 a0.2-mLaliquot of the original cell suspension to a small glass or plastic tube (e.g.,2- or 5-mLvolume). Chill on ice.
(3) Gently add0.4 mLice-cold cell permeabilizing solution. Wait15 s, keeping cells on ice.
(4) Gently add1.2 mLice-cold Acridine Orange base staining solution. Keep cells on ice in the dark.
(5) Measure and record cell fluorescence in the flow cytometer during the2 to 10 minafter 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~10⁶cells/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~10⁶cells/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~10⁶cells/mL.
(2) With a Pasteur pipet transfer1 mLcell suspension to a15-mLconical glass tube containing10 mLice-cold 70% ethanol. Fix cells≥2 hon ice.
(3) Centrifuge tubes5 minat 300 × g, 4℃. Remove all ethanol, rinse cells once with ice-cold PBS, and suspend in ice-cold PBS at a density of< 2 × 10⁶cells/mL.
(4) Withdraw0.2 mLcell suspension (≤ 2 × 10⁵cells) and transfer to a small tube (e.g.,2 or 5 mLvolume). Chill on ice.
(5) Add0.4 mLice-cold permeabilizing solution. Wait15 s, keeping cells on ice.
(6) Add1.2 mLice-cold Acridine Orange base staining solution. Keep cells on ice.
(7) Measure and record cell fluorescence in the flow cytometer during the2 to 10 minafter addition of Acridine Orange base staining solution.

265.35

C₁₇H₁₉N₃

494-38-2

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.