碧云天生物技术-Mito-Tracker Deep Red FM (线粒体深红荧光探针)(C1032-50μg)

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试剂> 细胞相关> 细胞标记与染色> 荧光染色

Mito-Tracker Deep Red FM (线粒体深红荧光探针)

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产品编号: C1032-50μg

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产品编号	产品名称	产品包装	产品价格
C1032-50μg	Mito-Tracker Deep Red FM (线粒体深红荧光探针)	50μg	418.00元
C1032-250μg	Mito-Tracker Deep Red FM (线粒体深红荧光探针)	250μg	1699.00元

Mito-Tracker Deep Red FM (线粒体深红荧光探针)，是一种具有细胞通透性、能够特异性地标记细胞中具有生物活性的线粒体，并检测线粒体膜电位的线粒体深红荧光探针。本产品为高纯度探针，可以兼容后续的细胞固定和通透，但染色后再固定和通透，荧光强度会有一定的下降。

本产品是一种深红荧光染料，只需简单地和细胞孵育，即可通过被动运输穿过细胞膜，并借助本探针含有的弱巯基反应性的氯甲基(mildly thiol-reactive chloromethyl)官能团特异性地标记有生物活性的线粒体。本探针含有的弱巯基反应性的氯甲基，可以和线粒体内蛋白的巯基反应并共价连接，因此后续实验使用多聚甲醛或甲醛等醛类固定剂，以及细胞通透的去垢剂Triton X-100等处理时，线粒体的荧光标记不会消失，但可能会有一定程度的下降(约下降2-8倍)。由于本荧光探针的激发光谱和发射光谱与常见的绿色和红色荧光探针通常不会重叠，因此非常适合用于多重荧光染色实验。

Mito-Tracker Deep Red FM分子式为C₃₄H₃₆Cl₂N₂，分子量为543.58，Mito-Tracker Deep Red FM为深红荧光探针，最大激发波长为644nm，最大发射波长为665nm。Mito-Tracker Deep Red FM的化学结构式以及激发光谱和发射光谱参考图1。

图1. Mito-Tracker Deep Red FM的化学结构式(A)及激发光谱和发射光谱(B)。

Mito-Tracker Deep Red FM可以用作线粒体特异性的荧光探针。和Rhodamine 123或JC-1相似，Mito-Tracker Deep Red FM对于线粒体的染色依赖于线粒体膜电位，因此该探针只能对活的细胞或组织进行染色，不能对固定或通透后的细胞或组织进行染色。使用Mito-Tracker Deep Red FM染色细胞线粒体效果参考图2。

图2. Mito-Tracker Deep Red FM (线粒体深红荧光探针)对于NRK-52E细胞(大鼠肾小管上皮细胞)的染色效果。Mito-Tracker Deep Red FM染色的NRK-52E细胞其线粒体呈现洋红色荧光(图B。注：为了区分Cy3、PE等普通红色荧光，深红荧光处理成洋红色伪彩)，Actin-Tracker Green-488 (C2201S)染色的NRK-52E细胞其微丝呈现绿色荧光(图C)，洋红色荧光、绿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 (C1027)染色。实测数据会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。注：Mito-Tracker Deep Red FM染色活细胞线粒体后经固定、通透处理，再进行Actin-Tracker Green-488染色和Hoechst 33342染核。

由于Mito-Tracker Deep Red FM在线粒体内的聚集依赖于线粒体的膜电位，因此本产品也可以作为线粒体膜电位的指示探针，并可以通过检测线粒体膜电位的变化来检测细胞凋亡。

按最终工作浓度为50-200nM计算，本产品每50µg可以配制约460-1800ml Mito-Tracker Deep Red FM工作液。

包装清单：

产品编号	产品名称	包装
C1032-50μg	Mito-Tracker Deep Red FM	50μg
C1032-250μg	Mito-Tracker Deep Red FM	50µg×5
—	说明书	1份

保存条件：

-20℃避光保存，至少两年有效。

注意事项：

Mito-Tracker Deep Red FM可以用于活细胞的线粒体荧光染色，不适合用于固定后细胞或者组织的染色；但在用本产品标记线粒体后，后续可以进行固定和通透，并用其它探针进行染色或进行免疫荧光染色，但染色后再固定和通透，荧光强度会有一定程度的下降。而且不同的细胞，固定和通透的影响会有所区别，建议对固定和通透试剂、条件进行一定的测试。

本产品仅包含非常微量的粉末，使用前请先离心数秒钟，使微量粉末充分沉降到管底。

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。

本产品对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。

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.Mito-Tracker Deep Red FM储存液的配制：
取一管50μg Mito-Tracker Deep Red FM粉末加入460μl的无水DMSO (anhydrous dimethylsulfoxide)，充分溶解后，得到浓度为200μM的Mito-Tracker Deep Red FM储存液。也可以根据需要配制成其它浓度的储存液，如1mM。适当分装后避光保存于-20℃或更低温度。
2.Mito-Tracker Deep Red FM工作液的配制：
a.取少量200μM Mito-Tracker Deep Red FM储存液按照1:1000-1:10,000的比例加入到细胞培养液或适当的溶液(例如含钙镁离子的HBSS)中，使最终浓度为20nM-200nM。例如取1µl 200μM的Mito-Tracker Deep Red FM储存液加入到1ml细胞培养液或适当的溶液(例如含钙镁离子的HBSS)中，混匀后即为200nM的Mito-Tracker Deep Red FM工作液。如果染色的细胞后续需要进行固定或通透等步骤，建议工作浓度为50nM-500nM。Hanks' Balanced Salt Solution (with Ca²⁺ & Mg²⁺) (C0219)可以向碧云天订购。
b.Mito-Tracker Deep Red FM工作液使用前需37℃预温育。
注：Mito-Tracker Deep Red FM工作液的浓度可以根据实际情况进行适当调整。为降低非特异性荧光染色和线粒体毒性，在染色效果可以接受的范围内，建议尽量使用较低浓度的Mito-Tracker Deep Red FM。
3.贴壁细胞的线粒体染色：
a.当细胞在细胞培养板或培养皿中培养至一定密度时，去除细胞培养液，加入步骤2中配制好的Mito-Tracker Deep Red FM工作液，37℃孵育15-30分钟。注：最佳孵育时间需根据细胞类型进行适当的优化。
b.去除Mito-Tracker Deep Red FM工作液，加入37℃预温育的新鲜细胞培养液。
c.用荧光显微镜、激光共聚焦显微镜或荧光酶标仪进行观察或检测。此时可观察到线粒体呈明亮的强荧光染色。如果染色效果欠佳，可以提高Mito-Tracker Deep Red FM工作液浓度或在推荐的时间范围内适当延长染色时间。
注：标记的Mito-Tracker Deep Red FM易淬灭，请注意尽快进行拍照等荧光检测。也可以通过降低荧光显微镜的激发光强度(即汞灯或LED光源)或适当降低Mito-Tracker Deep Red FM工作液浓度以延缓淬灭。
4.悬浮细胞的线粒体染色：
a.1000×g离心5分钟，弃上清，用37℃预热的Mito-Tracker Deep Red FM工作液轻轻重悬细胞，37℃孵育15-30分钟。注：最佳孵育时间需根据细胞类型进行适当的优化。
b.孵育结束后，1000×g离心5分钟，弃上清，加入37℃预温育的新鲜细胞培养液重悬细胞。
c.用荧光显微镜、激光共聚焦显微镜、流式细胞仪或荧光酶标仪进行观察、分析或检测。
注：如果需要在载玻片或盖玻片上固定悬浮细胞，可先用碧云天的Poly-D-lysine/多聚赖氨酸(ST508)处理载玻片或盖玻片。
5.染色后的细胞固定或细胞通透(选做)：
用本试剂染色线粒体后，可进行固定和通透，并用其它探针进行复染或进行其它的免疫荧光染色。
a.细胞固定：在染色后，用37℃温育的新鲜细胞培养液或HBSS洗涤细胞，小心吸除洗涤用的培养液，加入4%多聚甲醛固定液(例如P0099)或含3.7%新鲜甲醛的完全培养液，室温固定15-30分钟。固定完成后用HBSS洗涤2-3次。
注：采用37℃预热的固定液并在37℃固定约15min，可更好地维持细胞形态。对于内皮细胞，在本染料染色之后再用含3.7%甲醛的完全培养液于37℃孵育15分钟有良好的固定效果。
b.细胞通透：将固定后的细胞加入碧云天生产的免疫染色通透液(Triton X-100) (P0096)、免疫染色强力通透液(P0095)或含约0.2% Triton X-100的PBS，室温孵育10-15分钟。然后用PBS洗涤。使用本探针染色活细胞后，再固定和通透的细胞内荧光效果参考图3。
注1：对于内皮细胞，在本染料染色之后再用含约0.2% Triton X-100的PBS室温孵育10分钟有良好的通透效果。
注2：也可用丙酮冰浴通透5分钟，然后用PBS洗涤。即使后续无需抗体标记，丙酮通透处理对降低背景信号也会有一定的帮助。
相关产品：

产品编号	产品名称	包装
C1031	CFDA SE (细胞增殖示踪荧光探针)	5mg
C1032	Mito-Tracker Deep Red FM (线粒体深红荧光探针)	50µg/250µg
C1033	Actin-Tracker Green (微丝绿色荧光探针)	0.2ml
C1034	Mito-Tracker Deep Red 633 (线粒体深红荧光探针)	50µg/250µg
C1035	Mito-Tracker Red CMXRos (线粒体红色荧光探针,高纯度)	50μg/250μg
C1036	DiI (细胞膜红色荧光探针)	10mg
C1038	DiO (细胞膜绿色荧光探针)	10mg
C1039-10mg	DiD (细胞膜远红荧光探针)	10mg
C1041	ER-Tracker Red (内质网红色荧光探针)	20μl
C1042S	ER-Tracker Green (内质网绿色荧光探针)	20μl
C1043	Golgi-Tracker Red (高尔基体红色荧光探针)	1mg
C1045S	Golgi-Tracker Green (高尔基体绿色荧光探针)	1mg
C1046	Lyso-Tracker Red (溶酶体红色荧光探针)	50μl
C1047S	Lyso-Tracker Green (溶酶体绿色荧光探针)	50μl
C1048	Mito-Tracker Green (线粒体绿色荧光探针)	50μg
C1049B	Mito-Tracker Red CMXRos (线粒体红色荧光探针)	50μg/250μg
C1050	Tubulin-Tracker Red (抗体法微管红色荧光探针)	40μl
C1051S	Tubulin-Tracker Green (抗体法微管绿色荧光探针)	40μl
C1053S	7-AAD细胞活力检测试剂盒	200次
C2201S	Actin-Tracker Green-488 (微丝绿色荧光探针)	0.2ml
C2203S	Actin-Tracker Red-555 (微丝红色荧光探针)	0.2ml
C2205S	Actin-Tracker Red-594 (微丝红色荧光探针)	0.2ml
C2207S	Actin-Tracker Red-Rhodamine (微丝红色荧光探针)	0.2ml
C2213S	微管绿色荧光染色试剂盒(活细胞染色用)	20-200次
C2215S	微管深红荧光染色试剂盒(活细胞染色用)	20-200次

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