323192-14-9 Cell-Tracker Orange CMTMR 活细胞示踪探针(橙色)

Cell-Tracker Green CMFDA;CellTracker™ Orange CMTMR;Cell movement 细胞运动;Cell location 细胞定位;Cell Tracker dye 细胞示踪探针;CAS NO:323192-14-9;

产品描述

Cell-Tracker荧光探针是用来监测细胞运动、定位、增殖、迁移、趋化和侵袭的优秀工具。

Cell-Tracker Orange CMTMR,英文全名:5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (mixed isomers),能自由穿透细胞膜进入细胞,在胞内转化生成不具细胞膜渗透性的反应产物。该产物几次传代都能良好的保留在活细胞。在细胞群内,染料只会转移到子代细胞,不会转移到邻近细胞。

Cell-Tracker Orange CMTMR特地设计使其至少72h(典型有3~6代)能展示荧光,此染料表现出理想的示踪特征:稳定、工作浓度下无毒性、良好保留在细胞,且在生理pH下呈明亮荧光。另外,Cell-Tracker Red CMTPX的激发和发射光谱(541/565nm)与绿色荧光蛋白GFP很好的分开,适用于多重标记(见附表1. Cell-Tracker荧光探针的光谱特征)。

产品特性

1)  CAS NO.:323192-14-9

2)  同义名:CellTracker™ Orange CMTMR;Xanthylium, 9-[2-carboxy-4(or 5)-[[4-(chloromethyl) benzoyl]amino]phenyl]-3,6-bis(dimethylamino)-, inner salt

3)  分子式:C32H28ClN3O4

4)  分子量:554.04 g/mol

5)  外观:紫色固体

6)  溶解性:溶于DMSO

7)  化学结构式: 

保存与运输方法

保存:-20℃避光干燥保存,至少2年有效。

运输:冰袋运输。

注意事项

  1. 荧光染料都存在淬灭的问题,保存和操作过程中注意避光。
  2. 避免使用含氨基和巯基的缓冲液。
  3. 为了您的安全和健康,请穿实验服并戴一次性手套操作。

应用示例

文献1)McKee AS et al.Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12): E1122-31. PMID: 23447566

操作方法(多光子显微镜):CD4 T cellswere isolated from OTII or B6 mice using the CD4 T-cell negative isolation kit andwere labeled with 20 μM CMTMR or 2 μM CFSE, washed three times, and injected i.v. into B6 or CD11c-YFP recipients. Twenty-four hours later, these recipient mice were immunized with 20 μg of AF647-labeled ova + 200 μg of Alhydrogel and 5 mg of either BSA or DNase. From 20–24 h following immunization, mice were killed and their draining popliteal LNs were surgically removed for imaging. 

文献2)Nikitina EY et al.Combination ofγ‐irradiation and dendritic cell administration induces a potent antitumor response in tumor‐bearing mice: Approach to treatment of advanced stage cancer. Int J Cancer. 2001 Dec 15;94(6):825-33. PMID: 11745485

操作方法(组织内细胞分布评估):DCs were labeled with 20 μM cell tracker orange (CMTMR) by incubation in RPMI 1640 at 37℃ for 30 min. Cells were then washed in PBS once, incubated for 30 min more and injected (107/mouse) intravenously (i.v.) or subcutaneously (s.c.) near the tumor site. Eighteen hours later lung, spleen, liver, superficial inguinal and popliteal LN as well as tumor tissue were taken out and snap‐frozen in tissue freezing media at−80°C. Frozen tissue sections were examined by fluorescence microscopy using a 540 nm filter. Labeled cells were counted in 10 fields at the total magnification ×200.

Fig. DC labeled with cell tracker CMTMR inside MethA sarcoma. Unlabeled (I) or CMTMR‐labeled (II, III) DCs (107) were injected i.v. (II) or s.c. (III) into MethA sarcoma‐bearing mice 3 hr after irradiation of the tumor with 10 Gy. Tumors were excised 18 hr later and cryostat sections were performed. Slides were analyzed by fluorescence microscopy using a 540‐nm filter. One of the representative fields with a total magnification of 200× is shown. 

使用方法

1.细胞准备

在合适的培养基内培养细胞。贴壁细胞可以在含盖玻片的培养皿内爬片生长,装入足量的生长培养基。

2.操作步骤

以下描述的是将染料加到培养细胞以及在荧光显微镜下成像的步骤。各种因素,比如将染料加载到细胞或组织,可能都需根据特定的细胞类型对某些条件做出修改。

探针的最佳染色浓度需根据用途来调整。建议刚开展实验需要测定至少1个10倍范围内的浓度。一般来说,长期染色(≥3天)或使用快速分裂的细胞需5-25µM的染料。对于短期实验(比如活力测定),使用低浓度染料(0.5-5µM)。为了维持正常的细胞形态和降低潜在的伪影,尽可能使用低浓度的染料。

2.1制备Cell-Tracker染色工作液

①开瓶前将产品从冰箱取出,放到室温使其回温至少20min。

②用高质量的无水DMSO溶解粉末使其浓度为10mM。例如,对1mgCell-Tracker Green CMFDA(Mw:464.86 g/mol)冻干粉,加入215µlDMSO充分溶解,即得到10mM母液。

③ 用无血清培养基稀释母液到0.5-25µM的工作浓度。预热染色工作液到37℃。

2.2悬浮细胞染色步骤

① 离心收集细胞,吸掉上清液。用预热的Cell-Tracker染色工作液轻轻的重悬细胞。

② 在适合特定细胞类型的生长条件下孵育15-45min。

③ 离心细胞,吸掉Cell-Tracker染色工作液。

④ 加入选择的培养基,将标记好的细胞分配到载玻片或到选择的培养器皿内。

⑤根据附表1选择合适激发和发射波长的滤片来进行成像检测。

2.3贴壁细胞染色步骤

① 吸走培养基。

② 轻轻加入预热的Cell-Tracker染色工作液。

③ 在适合特定细胞类型的生长条件下孵育15-45min。

④吸掉Cell-Tracker染色工作液。

⑤ 加入选择的培养基。

⑥根据表1选择合适激发和发射波长的滤片来进行成像检测。

3.荧光显微镜观察

Cell-Tracker荧光探针可用带标准光学和图像增强的各种落射荧光光学显微镜检测。根据染料选择合适的滤片。见附表1 Cell-Tracker荧光探针的光谱特征。

相关产品

名称 规格                  

           

Cell-Tracker Green CMFDA 活细胞示踪探针(绿色) 1mg

Cell-Tracker Orange CMTMR 活细胞示踪探针(橙色)        

2×50μg

Cell-Tracker Red CMTPX 活细胞示踪探针(红色)

2×50μg

Fluorescein diacetate (FDA) 二乙酸荧光素

1g

Propidium Iodide 碘化丙啶(粉末)

10mg