7-Aminoactinomycin D (7-AAD) is a fluorescent DNA dye that is commonly used to detect or exclude non-viable cells in flow cytometry analysis because 7-AAD is usually excluded by living cells[1][ 2]. 7-AAD exhibits excitation spectra at 488, 546, and 578 nm and an emission spectrum at 650 nm [3]. Since 7-AAD is detected in the far-red range of the spectrum, it has minimal spectral overlap with commonly used probes and thus can be used in combination with probes such as FITC [1] [3]. 7-AAD has been used to assess apoptosis and cell-mediated cytotoxicity and to stain DNA in cells fixed and permeabilized by various methods [1] [2] [4].
7-AAD is a fluorescent derivative of actinomycin D that selectively binds to the GC region of DNA [5]. Taking advantage of the permeability of the cell membrane and the fact that the fluorescence intensity of 7-AAD is lower in early apoptotic cells and higher in late apoptotic and dead cells, it can be performed in fixed/permeabilized cells by flow cytometry. cycle analysis. Low concentrations (0.5-5μg/ml) of 7-AAD are often used to stain and exclude dead cells in flow cytometry; higher concentrations (10-20μg/ml) of 7-AAD are used to distinguish live cells (7-AAD negative ), apoptotic cells (7-AAD dim bright) and dead cells (7-AAD)[5].
References:
[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)
[5].Nadine C L Zembruski, et al. 7-Aminoactinomycin D for apoptosis staining in flow cytometry. 2012 Oct 1;429(1):79-81. doi: 10.1016/j.ab.2012.07.005. Epub 2012 Jul 14.
7-Aminoactinomycin D (7-AAD)是一种荧光DNA染料，通常用于检测或排除流式细胞术分析中的非活细胞，因为7-AAD通常被活细胞排除[1] [2]。7-AAD显示488、546和578 nm的激发光谱和650 nm的发射光谱[3]。由于在光谱的远红色范围内检测到7-AAD，因此它与常用探针的光谱重叠最小，因此可以与FITC等探针结合使用[1] [3]。7-AAD已被用于评估细胞凋亡和细胞介导的细胞毒性，并对通过多种方法固定和透化的细胞中的DNA进行染色[1] [2] [4]。
7-AAD是放线菌素D的荧光衍生物，选择性地与DNA的GC区域结合[5]。利用细胞膜的通透性以及7-AAD荧光强度在早期凋亡细胞中较低，而在晚期凋亡和死细胞中较高的特点，可以通过流式细胞术在固定/透化细胞中进行细胞周期分析。低浓度（0.5-5μg/ml）的7-AAD常用于流式细胞术中染色和排除死细胞；较高浓度（10-20μg/ml）的7-AAD被用于区分活细胞（7-AAD negative ）、凋亡细胞（7-AAD dim bright）和死细胞（7-AAD）[5]。