7-Aminoactinomycin D (7-AAD)
(Synonyms: 7-氨基放线菌素D; 7-AAD) 目录号 : GC33577A fluorescent DNA dye
Cas No.:7240-37-1
Sample solution is provided at 25 µL, 10mM.
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- Purity: >95.00%
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7-Aminoactinomycin D (7-AAD) is a fluorescent DNA dye that is commonly used for the detection or exclusion of non-viable cells in flow cytometric analysis, as it is generally excluded by live cells.1,2 It displays excitation spectra of 488, 546, and 578 nm and an emission spectrum of 650 nm.3 As 7-AAD is detected in the far red range of the spectrum, it exhibits minimal spectral overlap with commonly used probes, therefore it can be used in conjunction with probes such as FITC.1,3 7-AAD has been used to evaluate apoptosis and cell-mediated cytotoxicity and to stain DNA in cells that have been fixed and permeabilized by a variety of methods.1,2,4
1.Schmid, I., Uittenbogaart, C.H., Keld, B., et al.A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometryJ. Immunol. Methods170(2)145-157(1994) 2.Coder, D.M.Assessment of cell viabilityCurr. Protoc. Cytom.Suppl. 15(1997) 3.Stokke, T., Holte, H., and Steen, H.B.In vitro and in vivo activation of B-lymphocytes: A flow cytometric study of chromatin structure employing 7-aminoactinomycin DCancer Res.48(23)6708-6714(1988) 4.Lecoeur, H., Février, M., Garcia, S., et al.A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicityJ. Immunol. Methods253(1-2)177-187(2001)
| Cas No. | 7240-37-1 | SDF | |
| 别名 | 7-氨基放线菌素D; 7-AAD | ||
| Canonical SMILES | O=C(N[C@H](C(N1[C@@](C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]2C)=O)=O)=O)([H])CCC1)=O)C(C)C)[C@H]2NC(C(C(C(OC3=C(C)C(N)=C4)=C5C)=NC3=C4C(N[C@@H]6C(N[C@H](C(N7[C@@](C(N(C)CC(N(C)[C@@H](C(C)C)C(O[C@@H]6C)=O)=O)=O)([H])CCC7)=O)C(C)C)=O)=O)=C(N)C5=O)=O | ||
| 分子式 | C62H87N13O16 | 分子量 | 1270.43 |
| 溶解度 | DMF: 10 mg/ml,DMF:PBS(pH 7.2)(1:1): 0.5 mg/ml,DMSO: 5 mg/ml | 储存条件 | -20°C, protect from light,unstable in solution, ready to use. |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.7871 mL | 3.9357 mL | 7.8714 mL |
| 5 mM | 0.1574 mL | 0.7871 mL | 1.5743 mL |
| 10 mM | 0.0787 mL | 0.3936 mL | 0.7871 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
7-Aminoactinomycin D for apoptosis staining in flow cytometry
Anal Biochem 2012 Oct 1;429(1):79-81.PMID:22796502DOI:10.1016/j.ab.2012.07.005.
7-Aminoactinomycin D (7-AAD) is a DNA dye that distinguishes viable, apoptotic, and late apoptotic/dead cells in flow cytometry. Several staining protocols using 7-AAD have been described, but data on the influence of the 7-AAD concentration on the readout are not available. Therefore, we compared the results obtained by staining with 1, 5, 10, and 20μg/ml 7-AAD for 20min with the PE-Annexin V Apoptosis Detection Kit and Cell Death Detection ELISA(PLUS) in lymphocytes and CEM human leukemia cells. The results showed that 7-AAD staining with 5, 10, and 20μg/ml, but not with 1μg/ml, is suitable for quantification of apoptosis in flow cytometry.
WDR45 contributes to neurodegeneration through regulation of ER homeostasis and neuronal death
Autophagy 2020 Mar;16(3):531-547.PMID:31204559DOI:10.1080/15548627.2019.1630224.
Mutations in the macroautophagy/autophagy gene WDR45 cause β-propeller protein-associated neurodegeneration (BPAN); however the molecular and cellular mechanism of the disease process is largely unknown. Here we generated constitutive wdr45 knockout (KO) mice that displayed cognitive impairments, abnormal synaptic transmission and lesions in several brain regions. Immunohistochemistry analysis showed loss of neurons in prefrontal cortex and basal ganglion in aged mice, and increased apoptosis in prefrontal cortex, recapitulating a hallmark of neurodegeneration. Quantitative proteomic analysis showed accumulation of endoplasmic reticulum (ER) proteins in KO mouse. At the cellular level, accumulation of ER proteins due to WDR45 deficiency resulted in increased ER stress and impaired ER quality control. The unfolded protein response (UPR) was elevated through ERN1/IRE1 or EIF2AK3/PERK pathway, and eventually led to neuronal apoptosis. Suppression of ER stress or activation of autophagy through MTOR inhibition alleviated cell death. Thus, the loss of WDR45 cripples macroautophagy machinery in neurons and leads to impairment in organelle autophagy, which provides a mechanistic understanding of cause of BPAN and a potential therapeutic strategy to treat this genetic disorder.Abbreviations: 7-ADD: 7-Aminoactinomycin D; ASD: autistic spectrum disorder; ATF6: activating transcription factor 6; ATG: autophagy-related; BafA1: bafilomycin A1; BCAP31: B cell receptor associated protein 31; BPAN: β-propeller protein-associated neurodegeneration; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CDIPT: CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase); DDIT3/CHOP: DNA-damage inducible transcript 3; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HIP: hippocampus; HSPA5/GRP78: heat shock protein family A (HSP70) member 5; KO: knockout; LAMP1: lysosomal-associated membrane 1; mEPSCs: miniature excitatory postsynaptic currents; MG132: N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal; MIB: mid-brain; MTOR: mechanistic target of rapamycin kinase; PCR: polymerase chain reaction; PFA: paraformaldehyde; PFC: prefrontal cortex; PRM: parallel reaction monitoring; RBFOX3/NEUN: RNA binding protein, fox-1 homolog [C. elegans] 3; RTN3: reticulon 3; SEC22B: SEC22 homolog B, vesicle trafficking protein; SEC61B: SEC61 translocon beta subunit; SEM: standard error of the mean; SNR: substantia nigra; SQSTM1/p62: sequestosome 1; TH: tyrosine hydroxylase; Tm: tunicamycin; TMT: tandem mass tag; TUDCA: tauroursodeoxycholic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling; UPR: unfolded protein response; WDR45: WD repeat domain 45; WT: wild type; XBP1: X-box binding protein 1.
MIR145-3p promotes autophagy and enhances bortezomib sensitivity in multiple myeloma by targeting HDAC4
Autophagy 2020 Apr;16(4):683-697.PMID:31242129DOI:10.1080/15548627.2019.1635380.
Multiple myeloma (MM) is an incurable plasma cell malignancy with poor survival. Autophagy, a stress-responsive catabolic process mediated by lysosomal activity, plays a crucial role in the pathophysiology of MM. Growing evidence has indicated that dysregulated microRNAs (miRNAs) are associated with the aberrant autophagy in various human cancers. However, to date, few miRNAs have been reported to directly modulate autophagy in the pathobiology of MM. In this study, we investigated the role of MIR145-3p (microRNA 145-3p) in MM, with focus on cellular processes autophagy and cell death. Our results provided evidence that downregulation of MIR145-3p expression was associated with disease progression in human MM. MIR145-3p triggered autophagic flux through direct targeting of HDAC4 (histone deacetylase 4) in MM cells, leading to enhanced apoptosis. Silencing HDAC4 recapitulated the effects of MIR145-3p, whereas enforced expression of HDAC4 abrogated the effects of MIR145-3p. Furthermore, we showed that suppression of HDAC4 by MIR145-3p resulted in upregulation of the pro-apoptotic protein BCL2L11 and caused MTORC1 inactivation, which in turn led to enhanced autophagy and cell death. Importantly, we demonstrated that MIR145-3p mimic could potentiate the anti-MM activity of bortezomib in both in vitro and in vivo experiments. Overall, our findings indicate that MIR145-3p exerted a tumor suppression function in MM by inducing autophagic cell death and suggest that MIR145-3p-based targeted therapy would represent a novel strategy for MM treatment.Abbreviations: 3-MA: 3-methyladenine; 3'-UTR: 3'-untranslated region; 7-AAD: 7-Aminoactinomycin D; ACTB: actin beta; ANXA5: annexin A5; ATG5: autophagy related 5; ATG7: autophagy related 7; B2M: beta-2-microglobulin; BAF: bafilomycin A1; BCL2L11: BCL2 like 11; Bort: bortezomib; CASP3: caspase 3; CCK-8: Cell Counting Kit-8; CQ: chloroquine; Ct: threshold cycle; ctrl: control; DAPI: 4',6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HDAC4: histone deacetylase 4; ISS: International Staging System; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; miRNAs: microRNAs; MIR145-3p: microRNA 145-3p; MM: multiple myeloma; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PCs: plasma cells; PFS: progression-free survival; qRT-PCR: quantitative reverse transcription PCR; RPS6KB1: ribosomal protein S6 kinase B1; SD: standard deviation; siRNA: small interfering RNA; SQSTM1: sequestosome 1; STV: starvation; TUBB: tubulin beta class I.
Actinomycin D and 7-Aminoactinomycin D binding to single-stranded DNA
Biochemistry 1991 Oct 1;30(39):9469-78.PMID:1892847DOI:10.1021/bi00103a012.
The potent RNA polymerase inhibitors actinomycin D and 7-Aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-Aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.
N7-Substituted 7-Aminoactinomycin D analogues. Synthesis and biological properties
J Med Chem 1978 Sep;21(9):958-61.PMID:722760DOI:10.1021/jm00207a021.
A series of N7-substituted 7-Aminoactinomycin D analogues with alkyl, aralkyl, and heteroaralkyl substituents was synthesized and their biological properties were studied. All of these analogues proved to be 22- to 28-fold less toxic than actinomycin D when tested against human lymphoblastic leukemia cells (CCRF-CEM) in vitro. Against the P388 mouse leukemia in vivo, most of the analogues had activity comparable to actinomycin D and one was significantly more active. The results show that substitutions of this kind do not interfere with the antitumor activity of actinomycin D and may be useful for the design of modified actinomycin D analogues with greater selectivity.