N3,N3,N6,N6-Tetramethylacridine-3,6-diamine

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].

Research Areas >> Others

Signaling Pathways >> Others >> Others

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 ∼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.

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

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

[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.

DATA ITEMS CITED :

49

MOLECULAR FORMULA :

C17-H19-N3

MOLECULAR WEIGHT :

265.39

WISWESSER LINE NOTATION :

T C666 BNJ EN1&1 MN1&1

HEALTH HAZARD DATA

ACUTE TOXICITY DATA

TYPE OF TEST :

LD50 - Lethal dose, 50 percent kill

ROUTE OF EXPOSURE :

Subcutaneous

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

250 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Administration onto the skin

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

6630 mg/kg

TOXIC EFFECTS :

Tumorigenic - equivocal tumorigenic agent by RTECS criteria Liver - tumors

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Subcutaneous

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

657 mg/kg/63W-I

TOXIC EFFECTS :

Tumorigenic - equivocal tumorigenic agent by RTECS criteria Blood - lymphoma, including Hodgkin's disease Tumorigenic - tumors at site of application

TYPE OF TEST :

Sex chromosome loss and nondisjunction

TYPE OF TEST :

Specific locus test

TYPE OF TEST :

Specific locus test

TYPE OF TEST :

Sperm Morphology

TYPE OF TEST :

Micronucleus test

TYPE OF TEST :

Specific locus test

TYPE OF TEST :

DNA inhibition

TYPE OF TEST :

Cytogenetic analysis

MUTATION DATA

TEST SYSTEM :

Bird - chicken

DOSE/DURATION :

1695 ug/kg

REFERENCE :

47JMAE "Cytogenetic Assays of Environmental Mutagens," Hsu, T.C., ed., Totowa, NJ, Allanheld, Osmun & Co., 1982 Volume(issue)/page/year: -,137,1982 *** REVIEWS *** IARC Cancer Review:Animal Inadequate Evidence IMEMDT IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. (WHO Publications Centre USA, 49 Sheridan Ave., Albany, NY 12210) V.1- 1972- Volume(issue)/page/year: 16,145,1978 IARC Cancer Review:Human No Adequate Data IMEMDT IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. (WHO Publications Centre USA, 49 Sheridan Ave., Albany, NY 12210) V.1- 1972- Volume(issue)/page/year: 16,145,1978 IARC Cancer Review:Group 3 IMSUDL IARC Monographs, Supplement. (WHO Publications Centre USA, 49 Sheridan Ave., Albany, NY 12210) No.1- 1979- Volume(issue)/page/year: 7,56,1987