描述

SYTOX Orange Nucleic Acid Stain (5 mM in DMSO) 死细胞橙色荧光核酸染料

产品关键词：

SYTOX Orange ；SYTOX Green；SYTO 9；碘化丙啶 PI；荧光增白剂28；Calcofluor White M2R；中性粒细胞胞外诱捕网（NETs）； 

产品信息

产品描述

SYTOX Orange核酸染料（SYTOX Orange Nucleic Acid Stain），是一种高亲和力的核酸染料，轻易渗透进入受损的细胞质膜，但不能穿透活细胞膜。探针只需简单孵育，使用氦氖激光器（He-Ne Laser）的543nm激光或其它520-550nm光源进行激发，死细胞的核酸则呈明亮的橙色荧光。结合以上特征，以及荧光增强＞500倍（与核酸结合）的优点，使其成为一种简单且定量的一步法死细胞指示剂，当用荧光显微镜、荧光光度计、荧光酶标板和流式细胞仪检测。与死细胞染料-碘化丙啶（PI，MX4205）相比，SYTOX Orange的发射波长更短，且光谱更匹配与罗丹明滤片设置。另外，SYTOX Orange有更高的摩尔吸光系数和更大的荧光增强（与DNA结合后），这些都表明SYTOX Orange是一款更高灵敏度的死细胞染料或核复染剂。

SYTOX Orange这一死细胞核酸染料可与蓝色和绿色荧光的表面标记联合使用，用于多指标分析。也能将SYTOX Orange与任一具细胞渗透性的核酸染料比如：Hoechst 33258，用于双色标记死细胞和活细胞。

本品以DMSO储存液形式提供，浓度为5mM。只需用合适的生理缓冲液稀释到工作浓度进行简单孵育即可。适用于哺乳动物细胞、细菌和酵母菌。

产品特性

1） 同义名：SYTOX Orange dye；SYTOX Orange死细胞染料；SYTOX Orange细胞核染料；

2） 外观：红色溶液

3） 荧光特征：Abs/E_m=547/570nm（与DNA结合），荧光产量为0.9（图1），需注意细菌或真核细胞中SYTOX Orange的光谱特征可能发生变化。

4）渗透性：不能进入活细胞

保存与运输方法

保存：-20℃避光干燥保存，至少1年有效。

运输：冰袋运输。

应用示例

文献来源1）Lee Y, Reilly B, Tan C, Wang P, Aziz M. Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation. Front Immunol. 2021 Dec 23;12:780210. doi: 10.3389/fimmu.2021.780210. PMID: 35003095; PMCID: PMC8732379.

实验目的：SYTOX Orange是常用的核酸染料用来检测胞外陷阱（extracellular traps，ETs)。

染色对象：THP-1细胞，染色剂量：SYTOX Orange（0.6 µM）

Fig 1. eCIRP induces extracellular trap formation in THP-1 cells. (A) THP-1 cells were treated with PBS or rmCIRP (1 µg/ml) in the presence of SYTOX Orange (0.6 µM) and subjected to the time-lapse live-cell imaging immediately after the treatment with rmCIRP. The upper panel shows the cells treated with PBS without rmCIRP, and the lower panel shows the cells treated with rmCIRP at different time points. Scale bar, 100 µm. (B) The dose-dependent effect of rmCIRP was quantified by counting the cells positive to SYTOX Orange. (C) Data shows the percentage of THP-1 cells positive to SYTOX Orange at 16 h after rmCIRP treatment (n = 4–5 microscopic fields for each condition). One-way ANOVA with Turkey method: *p <0.05. (D) THP-1 cells were pre-treated with IgG isotype Abs (10 µg/ml) or anti-TLR4 Abs (10 µg/ml) for 30 min. These cells were then treated with PBS or rmCIRP (0.5 µg/ml) in the presence of SYTOX Orange (0.6 µM) and subjected to the time-lapse live-cell imaging immediately after the treatment with rmCIRP. The time-lapse live-cell imaging data were acquired up to 18 h. The representative microscopic images taken at 18 h of rmCIRP treatment are captured and shown. Scale bar, 100 µm. (E) The percentages of SYTOX Orange positive cells in various experimental groups with time are shown.

文献来源2）Katarzyna Dubiel, Camille Henry, Lisanne M Spenkelink, Alexander G Kozlov, Elizabeth A Wood, Slobodan Jergic, Nicholas E Dixon, Antoine M van Oijen, Michael M Cox, Timothy M Lohman, Steven J Sandler, James L Keck, Development of a single-stranded DNA-binding protein fluorescent fusion toolbox, Nucleic Acids Research, Volume 48, Issue 11, 19 June 2020, Pages 6053–6067, https://doi.org/10.1093/nar/gkaa320

实验目的：使用SYTOX Orange实时监测dsDNA。

实验方法：Double-stranded DNA was visualized in real time by staining with 150 nM SYTOX Orange excited by a 568-nm laser (Coherent, Sapphire 568–200 CW) at 150 μW/cm2. The SSB–GFP was excited at 700 μW/cm2 with a 488 nm laser (Coherent, Sapphire 488–200 CW). Imaging was done with an EMCCD camera (Photometics, Evolve 512 Delta). The analysis was done with ImageJ using in-house built plugins. The rate of replication of a single molecule was obtained from its trajectory and calculated for each segment that has constant slope.

Fig 2. Single-molecule rolling-circle replication assays. (B) Kymograph of an individual DNA molecule undergoing leading- and lagging-strand replication. The gray indicates the fluorescence intensity of dsDNA stained by SYTOX orange.

染色流程（更多内容见说明书）

以下步骤适用于任何细胞类型，需注意不同细胞类型所需的探针工作浓度范围有差异（见表2）。生长培养基、细胞密度、是否存在其它细胞、和其它因素都可能影响染色。总的来说，不在磷酸盐的缓冲液中能得到最好的结果。玻璃器皿上残留的去污剂也有可能影响许多有机体的真实染色，导致含或不含细胞的溶液中都能发现明亮荧光的材料。确保用温和去污剂来清洗玻璃器皿，用热自来水完全冲洗干净，最后用去离子水清洗数次。

注意事项

• 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
• 为了您的安全和健康，请穿实验服并戴一次性手套操作。

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