吖啶橙

吖啶橙基是一种细胞渗透性荧光染料,可将生物体(细菌、寄生虫、病毒等)染色为亮橙色,并在适当条件下使用(pH=3.5,Ex=460 nm),将人类细胞区分为绿色,以便通过荧光显微镜进行检测。吖啶橙基与dsDNA(Ex=488,Em=520-524)结合时发出绿色荧光,与ssDNA(Ex=457,Em=630-644)或ssRNA(Ex=457,Em=630-444)结合时发红色荧光,也可用于细胞周期分析[1][2][3]。

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指南(以下是我们推荐的方案。该方案仅提供指南,应根据您的具体需要进行修改)。1.未固定细胞的DNA和RNA的差异染色[1]：(1)使用发射滤光片和分色镜,在488nm处设置激发流式细胞仪,区分绿色荧光(在515-545nm处测量)和红色发光(最好在640或650nm以上测量)。(2) 将0.2-mL等分的原始细胞悬浮液转移到小玻璃或塑料管中(例如,2-mL或5-mL体积)。在冰上冷却。(3) 轻轻加入0.4 mL冰冷电池渗透溶液。等待15秒,使电池保持在冰上。(4) 轻轻加入1.2 mL冰镇吖啶橙染色液。在黑暗中将电池放在冰上。(5) 添加吖啶橙碱性染色液后2至10分钟内,在流式细胞仪中测量并记录细胞荧光。2.固定细胞的差异染色[1]：(1a)对于悬浮培养或血液样本中的细胞：用冰凉PBS冲洗细胞一次,然后在∼106个细胞/mL。(1b)对于附着在组织培养板上的细胞：用胰酶消化法从烧瓶或培养板中收集细胞,将胰酶消化细胞与悬浮在培养基中的细胞(主要是分离的有丝分裂细胞和死细胞)混合,并用含有血清的培养基冲洗一次,使胰酶失活。将电池悬挂在冰冷的PBS中∼106个细胞/mL。(1c)对于从实体肿瘤中分离出来的细胞：冲洗不含任何用于细胞分离的酶的细胞,并将其悬浮在冰冷的PBS中∼106个细胞/mL。(2) 用巴斯德移液管将1mL细胞悬液转移到含有10mL冰凉70%乙醇的15mL锥形玻璃管中。固定单元格≥冰上2小时。(3) 离心管在300×g,4℃下5分钟。去除所有乙醇,用冰凉PBS冲洗细胞一次,并以<2×106个细胞/mL的密度悬浮在冰凉的PBS中。(4) 提取0.2 mL细胞悬液(≤ 2×105个细胞),然后转移到一个小管中(例如,2或5 mL体积)。在冰上冷却。(5) 添加0.4 mL冰凉渗透溶液。等待15秒,使电池保持在冰上。(6) 添加1.2 mL冰镇吖啶橙基染色液。将电池放在冰上。(7) 添加吖啶橙碱性染色液后2至10分钟内,在流式细胞仪中测量并记录细胞荧光。

[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