ER-Tracker Green

ER-Tracker Green是一种具有细胞膜通透性的内质网(endoplasmic reticulum, ER)绿色荧光探针，对内质网具有高度选择性，可以用于活细胞内质网特异性荧光染色。ER-Tracker Green为采用Molecular Probes公司的BODIPY® FL进行了荧光标记的glibenclamide。Glibenclamide即glyburide，中文名称为格列本脲，是一种常用2型糖尿病的治疗药物，可以高度结合主要定位在内质网上的包含ATP敏感的钾离子通道(KATP channel)的磺脲类(sulphonylurea)受体。因此荧光标记的glibenclamide就可以用作内质网特异性的荧光探针。ER-Tracker Green适用于活细胞内质网的荧光染色，但不适合用于固定细胞内质网的荧光染色。使用本产品染色活细胞内质网的效果参考图1。图1.ER-Tracker Green (内质网绿色荧光探针)对于NRK-52E细胞(大鼠肾小管上皮细胞)的染色效果。ER-Tracker Green染色的NRK-52E细胞其内质网呈现绿色荧光(图B)，Mito-Tracker Red CMXRos 染色的NRK-52E细胞其线粒体呈现红色荧光(图C)，绿色荧光、红色荧光及细胞核蓝色荧光的叠加(merge)效果见图D。其中细胞核使用Hoechst 33342 染色。本图仅作参考，实际检测效果会因实验条件、检测仪器等的不同而存在差异。ER-Tracker Green的分子式为C37H42BClF2N6O6S，分子量为783.1。ER-Tracker Green呈绿色荧光，检测时最大激发光波长为504nm，最大发射光波长为511nm。ER-Tracker Green的化学结构式和激发、发射光谱图参考图2。ER-Tracker Green对于细胞的毒性极低，而传统的DiOC6(3)对ER染色的同时也对细胞有一定的毒性。本产品包装中提供了ER-Tracker Green稀释液，使ER-Tracker Green的使用更加便捷。按照1:1000的比例稀释，每10µl可以配制10ml ER-Tracker Green工作液；按照1:3000的比例进行稀释，每10µl可以配制30ml ER-Tracker Green工作液。 注意事项 ： ER-Tracker Green (1mM)在4℃、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25℃水浴温育片刻至全部融解后使用。对于微量的液体，每次使用前先离心数秒钟，使液体充分沉降到管底。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。格列本脲的药理学活性可能会影响内质网的一些功能；某些特殊细胞中磺脲类受体的可变表达可能会导致非内质网特异性染色。ER-Tracker Green适用于活细胞内质网的荧光染色，但不适合用于固定细胞内质网的荧光染色。如果经ER-Tracker Green染色后的细胞需要进行固定操作，使用4%多聚甲醛在37℃固定2分钟。ER-Tracker Green染色后的细胞不能用Triton X-100通透，Triton X-100通透处理会导致ER-Tracker Green的荧光染色消失。需自备盖玻片和载玻片(可以向阿拉丁订购)。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.ER-Tracker Green工作液的配制： a.取少量ER-Tracker Green按照1:1000的比例加入到ER-Tracker Green稀释液中。例如取1µl ER-Tracker Green加入到1ml ER-Tracker Green稀释液中。混匀后即为ER-Tracker Green工作液。 b.ER-Tracker Green工作液使用前需37℃预温育。 注：工作液中ER-Tracker Green的浓度可以根据实际情况进行适当调整，推荐的稀释比例调整范围为1:1000-1:3000。为降低背景，在染色效果可以接受的范围内，建议尽量使用较低浓度的ER-Tracker Green。 2.内质网的荧光标记： a.去除细胞培养液，用适量的溶液如HBSS with Ca2+ & Mg2+ (Hanks’ Balanced Salt Solution with Ca2+ & Mg2+)洗涤生长在盖玻片上的细胞。注：HBSS with Ca2+ & Mg2+ 可以向阿拉丁订购；对于悬浮细胞的染色可以参考贴壁细胞的染色方法进行。 b.去除洗涤液，加入步骤1配制好的并37℃预温育的ER-Tracker Green染色工作液，与细胞37℃共孵育15-30分钟。 c.去除ER-Tracker Green染色工作液，用细胞培养液洗涤细胞1-2次。 d.随后通常用荧光显微镜或激光共聚焦显微镜进行观察。此时可观察到内质网呈明亮的强荧光染色。 e.如果经ER-Tracker Green染色后的细胞需要进行固定，可以使用4%甲醛37℃固定2分钟。固定后用适当的洗涤液洗涤2-3次，每次5分钟，随后可以进行复染或滴加适当的抗荧光淬灭封片液，最后封片观察。注意：ER-Tracker Green染色的细胞不能用Triton X-100通透，Triton X-100通透处理会导致ER-Tracker Green的荧光染色消失。 Precautions ： ER-Tracker Green (1mM) will solidify on ice or at lower temperatures such as 4℃ and stick to the bottom, walls or lid of the centrifuge tube. It should be thawed completely in a water bath at 20-25℃ prior to use. For trace amounts of liquid, centrifuge briefly before each use to collect the liquid at the bottom of the tube.Fluorescence intensity decays. Avoid light to minimize the quenching of fluorescence.The pharmacological activity of glibenclamide may affect some functions of the ER. Variable expression of sulfonylurea receptors in some specific cells may result in non-ER-specific staining.ER-Tracker Green is suitable for staining ER in live cells, but not ER in fixed cells. When cells stained with ER-Tracker Green need to be fixed, fix cells with 4% paraformaldehyde at 37℃ for 2 minutes.ER-Tracker Green stained cells cannot be permeabilized with Triton X-100 which will result in the loss of ER-Tracker Green fluorescent staining.Coverslips and slides are required but not supplied. They can be ordered from .This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1.Preparation of ER-Tracker Green working solutiona. Dilute an appropriate amount of ER-Tracker Green (1mM) in the ER-Tracker Green Dilution Buffer at a ratio of 1:1000. For example, mix 1µl of ER-Tracker Green with 1ml of ER-Tracker Green Dilution buffer to obtain the ER-Tracker Green working solution.b.Prewarm the ER-Tracker Green working solution at 37℃ before use.Note: The concentration of ER-Tracker Green in the working solution can be adjusted within the range of 1:1000-1:3000 based on the staining results. To minimize the staining background, a lower concentration of ER-Tracker Green is preferred as long as an acceptable staining result can be obtained.2.Fluorescent labeling of ERa. Remove the cell culture medium and wash cells grown on coverslips with an appropriate amount of solution such as Hanks' Balanced Salt Solution with Ca2+ & Mg2+ (, C0219). For staining of suspension cells, centrifuge to collect cells and refer to the staining method for adherent cells.b.Remove the wash buffer, add the ER-Tracker Green working solution pre-incubated at 37℃, and incubate at 37℃ for 15-30 min.c. Remove the ER-Tracker Green working solution and wash cells 1-2 times with cell culture medium.d.Examine cells by fluorescence microscope or confocal microscope. e. If the ER-Tracker Green stained cells need to be fixed, fix with 4% paraformaldehyde for 2 min at 37℃. After fixation, wash cells 2-3 times with appropriate wash buffer for 5 minutes each, and then counterstain cells or mount cells with antifade mounting medium. Note: ER-Tracker Green stained cells cannot be permeabilized with Triton X-100 which will result in the loss of ER-Tracker Green fluorescent staining.

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