ER-Tracker Red (内质网红色荧光探针)

ER-Tracker Red

E745770-20μl

20μl

避光,-20°C储存

超低温冰袋运输

-20℃保存，半年有效。ER-Tracker Red需-20℃避光保存。

ER-Tracker Red是一种具有细胞膜通透性的内质网(endoplasmic reticulum, ER)红色荧光探针，对内质网有高度选择性，可以用于活细胞内质网特异性荧光染色。ER-Tracker Red为采用Molecular Probes公司的BODIPY TR进行了荧光标记的glibenclamide。Glibenclamide即glyburide，中文名为格列苯脲，是一种2型糖尿病治疗药物，可以结合主要定位在内质网上的包含ATP敏感的钾离子通道(KATPchannel)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 Red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。ER-Tracker Red的分子式为C44H42BClF2N6O7S2，分子量为915.23，ER-Tracker Red的化学结构式参考图1。 图1.ER-Tracker Red的化学结构式。 ER-Tracker Red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。ER-Tracker Red呈红色荧光，检测时的最大激发波长为587nm，最大发射波长为615nm。ER-Tracker Red的激发光谱和发射光谱参考图2。 图2.ER-Tracker Red的激发光谱和发射光谱。 ER-Tracker Red按照后附的使用说明染色后，用甲醛等固定后染色效果可以被部分保留。ER-Tracker Red在活细胞中的染色效果参考图2。 图3.ER-Tracker Red(内质网红色荧光探针)对于NRK-52E细胞(大鼠肾小管上皮细胞)的染色效果。Mito-Tracker Green染色的NRK-52E细胞其线粒体呈现绿色荧光(图B)，ER-Tracker Red染色的NRK-52E细胞其内质网呈现红色荧光(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。提供了ER-Tracker Red 稀释液，使ER-Tracker Red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 Red工作液；按照1:3000的比例稀释，每10μl可以配制30ml ER-Tracker Red工作液。 注意事项 ： ER-TrackerRed(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。需自备盖玻片和载玻片(可以向阿拉丁订购)。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 Red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 Red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 Red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 Red的荧光染色消失。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 Red工作液的配制：a.取少量ER-Tracker Red按照1:1000的比例加入到ER-Tracker Red 稀释液中。例如取1μl ER-Tracker Red加入到1ml ER-Tracker Red 稀释液中。混匀后即为ER-Tracker Red工作液。b.ER-Tracker Red工作液使用前需37℃预温育。注：工作液中ER-Tracker Red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 Red。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 Red染色工作液，与细胞37℃共孵育15-30分钟。c.去除ER-Tracker Red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 Red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 Red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 Red的荧光染色消失。 Precautions ： ER-Tracker Red (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.Coverslips and slides are required but not supplied. They can be ordered from .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 Red is suitable for staining ER in live cells, but not ER in fixed cells. When cells stained with ER-Tracker Red need to be fixed, fix cells with 4% paraformaldehyde at 37℃ for 2 minutes.ER-Tracker Red stained cells cannot be permeabilized with Triton X-100 which will result in the loss of ER-Tracker Red fluorescent staining.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 Red working solutiona.Dilute an appropriate amount of ER-Tracker Red (1mM) in the ER-Tracker Red Dilution Buffer at a ratio of 1:1000. For example, mix 1µl of ER-Tracker Red with 1ml of ER-Tracker Red Dilution buffer to obtain the ER-Tracker Red working solution.b.Prewarm the ER-Tracker Red working solution at 37℃ before use.Note: The dilution ratio of ER-Tracker Red 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 Red 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 Red working solution pre-incubated at 37℃, and incubate at 37℃ for 15-30 min.c. Remove the ER-Tracker Red 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 Red 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 an appropriate antifade mounting medium. Note: ER-Tracker Red stained cells cannot be permeabilized with Triton X-100 which will result in the loss of ER-Tracker Red fluorescent staining.