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Acridine Orange base
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Acridine Orange base
CAS No. : 494-38-2
MCE 站:Acridine Orange base
产品活性:Acridine Orange base 是一种细胞通透性荧光染料,能将生物体 (细菌,寄生虫,病毒等) 染成亮橙色,而当在适当条件下 (pH=3.5, Ex=460 nm) 可将人类细胞区别性地染成绿色,以便用荧光显微镜进行检测。Acridine Orange base 与 dsDNA 结合时发出绿色荧光 (Ex=488, Em=520-524),与 ssDNA (Ex=457, Em= 630-644) 或 ssRNA 结合时发出红色荧光 (Ex=457, Em=630-644),也可用于细胞周期测定。
研究领域:Others
作用靶点:Others
In Vitro: 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) 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:
(1a) For cells in suspension culture or hematologic samples: Rinse cells once with ice-cold PBS and suspend in ice-cold PBS at 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 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 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℃. 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.
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