ER-Tracker Green
目录号 : GC64788
ER-Tracker Green 是一种内质网特异性的荧光染料。
Cas No.:730931-46-1
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1.制备染色液
(1)配置DMSO储存液:使用DMSO溶解ER-Tracker Green，配置浓度为1mM的储存液。
注意:未使用的储存液分装后在-20℃或-80°C避光保存，避免反复冻融。
(2)配置工作液:用合适的缓冲液(如:无血清培养基或PBS)稀释储存液，配制浓度为100 nM-1 μM的工作液。
注意:请根据实际情况调整工作液浓度，现用现配。
2.细胞悬浮染色
(1)悬浮细胞：经4°C、1000g离心3-5分钟，弃去上清液，用PBS清洗两次，每次5分钟。
(2)贴壁细胞：使用PBS清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
(3)加入1mL的ER-Tracker Green工作溶液重悬约106个细胞，室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(4)孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
(5)用预温的无血清细胞培养基或PBS重悬细胞。通过荧光显微镜或流式细胞术观察。
3.细胞贴壁染色
(1)在无菌盖玻片上培养贴壁细胞。
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
(3)从盖玻片的一角加入100μL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。
(4)室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(5)孵育结束后吸弃染料工作液，使用预温的培养液清洗盖玻片2~3次。
4.显微镜检测：ER-Tracker Green的最大激发光/发射光分别为489/520 nm。
注意事项：
① 如果要固定染色的细胞，建议使用4%甲醛在37℃下固定2分钟；
② 如果染色细胞需要通透，建议使用ER–Tracker Blue–White DPX（GB30171）；
③ 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。
ER-Tracker Green is a fluorescent dye that specific for endoplasmic reticulum[1].
ER tracker Green (1 mM, incubated in OptiMEM for 30 min at 37 °C) is used for endoplasmic reticulum staining[1].
[1]. J Jacob Strouse, et al. Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters. Anal Biochem. 2013 Jun 1;437(1):77-87.
| Cas No. | 730931-46-1 | SDF | Download SDF |
| 分子式 | C37H42BClF2N6O6S | 分子量 | 783.09 |
| 溶解度 | DMSO : 50 mg/mL (63.85 mM; Need ultrasonic) | 储存条件 | Store at -20°C,protect from light |
| 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 | 1.277 mL | 6.385 mL | 12.7699 mL |
| 5 mM | 0.2554 mL | 1.277 mL | 2.554 mL |
| 10 mM | 0.1277 mL | 0.6385 mL | 1.277 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。
In Situ Partial Treatment of Single Cells by Laminar Flow in the "Open Space"
Anal Chem 2019 Jan 15;91(2):1644-1650.PMID:30558412DOI:10.1021/acs.analchem.8b05313.
Regional difference of a single cell is nonignorable, which means it is not precise to investigate the single cell as a homogeneous object. A convenient method to investigate cellular response to the treatment at the subcellular level is still needed. In this work, we developed a microfluidic approach for manipulating a partial region of a single adherent cell by generating a stable distribution of microenvironments. By controlling flow rates and the gap between the probe and substrate, the diffusion effect can be adjusted as needed. Distribution of the solute was revealed by fluorescein, demonstrating the stability of the interface between two miscible fluids. Partial lysis of single cells (Caco-2, MCF-7, U87 cells) was successfully performed, and partial staining (Mito-Tracker Green FM, Mito-Tracker Red CMXRos, ER-Tracker Green) of a single cell (U87) was explored. Self-healing and regeneration of a single U87 cell after partial lysis was also observed. All of those results demonstrated the feasibility and significance of our idea. The method would be a promising tool for further single-cell analysis and subcellular research.
[Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes]
Zhejiang Da Xue Xue Bao Yi Xue Ban 2019 May 25;48(1):65-74.PMID:31102360DOI:10.3785/j.issn.1008-9292.2019.02.11.
Objective: To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). Methods: Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. Results: Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P<0.01). Conclusions: Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.
Application of proteomics to identify the target molecules involved in Lonicera japonica-induced photokilling in human lung cancer CH27 cells
BMC Complement Altern Med 2013 Oct 1;13:244.PMID:24083475DOI:10.1186/1472-6882-13-244.
Background: The Lonicera japonica has been used as natural and healthy drink for its anti-inflammatory effect and pleasant odor in China and Taiwan. Methods: 2D electrophoresis was used to analyze the proteins involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis. The fluorescent dyes MitoTracker Red CMXRos, calcein AM and JC-1 were used to elucidate mitochondrial function. The protein expression was performed by Western blotting. Fluorescent image of endoplasmic reticulum was accomplished by using ER-Tracker Green. This study used fluorescent dye CM-H2DCFDA to detect intracellular generation of reactive oxygen species. Results: The identified proteins can be classified into three major groups, which include proteins involved in mitochondrial function, cytoskeleton-related proteins and proteins associated with endoplasmic reticulum (ER) stress. Photoactivated Lonicera japonica caused a significant effect on the mitochondrial function and ER stress in CH27 cells. The reactive oxygen species producing was found to be involved in photoactivated Lonicera japonica-induced CH27 cell apoptosis. Conclusion: Mitochondria and endoplasmic reticulum are the integral targets in photoactivated Lonicera japonica-induced CH27 cell apoptosis. We also demonstrated that ethyl acetate fraction of Lonicera japonica extracts caused photocytotoxicity in a dose-dependent manner in CH27 cells. This could explain the fact that the ethyl acetate fraction of Lonicera japonica extracts may contain compounds which exhibit the photosensitizing activity in CH27 cells.