F2611708计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存,干燥。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 2 篇同行评审文献引用。
概述
产品组成
产品名称 | 包装 |
JC-1(200×) | 100μL/管，共5管 |
超纯水 | 90mL |
JC-1染色缓冲液(5×) | 80mL |
CCCP(10mM) | 20μL |
产品简介
线粒体膜电位检测试剂盒（JC-1）是一种以JC-1为荧光探针，快速灵敏地检测细胞、组织或纯化的线粒体膜电位变化的试剂盒，可以用于早期的细胞凋亡检测。
JC-1是一种广泛用于检测线粒体膜电位△Ψm 的理想荧光探针。可以检测细胞、组织或纯化的线粒体膜电位。在线粒体膜电位较高时，JC-1聚集在线粒体的基质中，形成聚合物，可以产生红色荧光；在线粒体膜电位较低时，JC-1不能聚集在线粒体的基质中，此时JC-1为单体，可以产生绿色荧光。这样就可以非常方便地通过荧光颜色的转变来检测线粒体膜电位的变化。常用红绿荧光的相对比例来衡量线粒体去极化的比例。
线粒体膜电位的下降是细胞凋亡早期的一个标志性事件。通过JC-1从红色荧光到绿色荧光的转变可以很容易地检测到细胞膜电位的下降，同时也可以用JC-1从红色荧光到绿色荧光的转变作为细胞凋亡早期的一个检测指标。
JC-1单体的最大激发波长为515nm，最大发射波长为529nm；JC-1聚合物的最大激发波长为585nm，最大发射波长为590nm。实际观察时，使用常规的观察红色荧光和绿色荧光的设置即可。
本试剂盒提供了CCCP作为诱导线粒体膜电位下降的阳性对照。
对于六孔板中的样品，本试剂盒共可以检测100个样品；对于12孔中的样品，本试剂盒共可以检测200个样品。
使用说明
1. JC-1染色工作液的配制
六孔板每孔所需JC-1染色工作液的量为1mL，其他培养器皿的JC-1染色工作液的用量以此类推：对于细胞悬液每50～100万细胞需0.5mL JC-1染色工作液。取适量JC-1（200×），按照每50μL JC-1（200×）加入8mL超纯水的比例稀释JC-1。剧烈震荡充分溶解并混匀JC-1。然后再加入2mL JC-1染色缓冲液（5×），混匀后即为JC-1染色工作液。
2. 阳性对照的设置
把试剂盒中提供的CCCP（10mM）推荐按照1∶1000的比例加入到细胞培养液中，稀释至10μM，处理细胞20分钟。随后按照下述方法装载JC-1，进行线粒体膜电位的检测。对于大多数细胞，通常10μM CCCP处理20分钟后线粒体的膜电位会完全丧失，JC-1染色后观察应呈绿色荧光；而正常的细胞经JC-1染色后应显示红色荧光。对于特定的细胞，CCCP的作用浓度和作用时间可能有所不同，需自行参考相关文献资料决定。
3. 对于悬浮细胞
取10～60万细胞，重悬于0.5mL细胞培养液中，细胞培养液中可以含血清和酚红。
加入0.5mL JC-1染色工作液，颠倒数次混匀。细胞培养箱中37℃孵育20分钟。
在孵育期间，按照每1mL JC-1染色缓冲液（5×）加入4mL蒸馏水的比例，配制适量的JC-1染色缓冲液（1×），并放置于冰浴。
37℃孵育结束后，600g 4℃离心3～4分钟，沉淀细胞。弃上清，注意尽量不要吸除细胞。
用JC-1染色缓冲液（1×）洗涤2次：加入1mL JC-1染色缓冲液（1×）重悬细胞，600g 4℃离心3～4分钟，沉淀细胞，弃上清。再加入1mL JC-1染色缓冲液（1×）重悬细胞，600g 4℃离心3～4分钟，沉淀细胞，弃上清。
再用适量JC-1染色缓冲液（1×）重悬后，用荧光显微镜或激光共聚焦显微镜观察，也可以用荧光分光光度计检测或流式细胞仪分析。
4. 对于贴壁细胞
注意：对于贴壁细胞，如果希望采用荧光分光光度计或流式细胞仪检测，可以先收集细胞，重悬后参考悬浮细胞的检测方法。
对于六孔板的一个孔，吸除培养液，根据具体实验如有必要可以用PBS或其它适当溶液洗涤细胞一次，加入1mL细胞培养液。细胞培养液中可以含有血清和酚红。
加入1mL JC-1染色工作液，充分混匀。细胞培养箱中37℃孵育20分钟。
在孵育期间，按照每1mL JC-1染色缓冲液（5×）加入4mL蒸馏水的比例，配制适量的JC-1染色缓冲液（1×），并放置于冰浴。
37℃孵育结束后， 吸除上清，用JC-1染色缓冲液（1×）洗涤2次。
加入2mL细胞培养液，培养液中可以含有血清和酚红。
荧光显微镜或激光共聚焦显微镜下观察。
5. 对于纯化的线粒体
把配制好的JC-1染色工作液再用JC-1染色缓冲液（1×）稀释5倍。
0.9mL 5倍稀释的JC-1染色工作液中加入0.1mL总蛋白量为10～100μg纯化的线粒体。
用荧光分光光度计或荧光酶标仪检测：混匀后直接用荧光分光光度计进行时间扫描（time scan），激发波长为485nm，发射波长为590nm。如果使用荧光酶标仪，激发波长不能设置为485nm时，可以在475～520nm范围内设置激发波长。另外，也可以参考下面步骤6中的波长设置进行荧光检测。
用荧光显微镜或激光共聚焦显微镜观察：方法同下面的步骤6。
6. 荧光观测和结果分析
检测JC-1单体时可以把激发光设置为490nm，发射光设置为530nm；检测JC-1聚合物时，可以把激发光设置为525nm，发射光设置为590nm。注意：此处测定荧光时不必把激发光和发射光设置在最大激发波长和最大发射波长。如使用荧光显微镜观察，检测JC-1单体时可以参考观察其它绿色荧光时的设置，如观察GFP或FITC时的设置；检测JC-1聚合物时可以参考观察其它红色荧光，如碘化丙啶或Cy3时的设置。出现绿色荧光说明线粒体膜电位下降，并且该细胞很可能处于细胞凋亡早期。出现红色荧光说明线粒体膜电位比较正常，细胞的状态也比较正常。
保存条件
-20℃避光保存，尽量避免反复冻融，有效期一年。
超纯水和JC-1染色缓冲液（5×）也可4℃保存。
注意事项
1. JC-1（200×）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。
2. 必须先把JC-1（200×）用试剂盒提供的超纯水充分溶解混匀后，才可以加入JC-1染色缓冲液（5×）。不可先配制JC-1染色缓冲液（1×）再加入JC-1（200×），这样JC-1会很难充分溶解，会严重影响后续的检测。
3. 装载完JC-1后用JC-1染色缓冲液（1×）洗涤时，使JC-1染色缓冲液（1×）保持4℃左右，此时的洗涤效果较好。
4. JC-1探针装载完并洗涤后尽量在30分钟内完成后续检测。在检测前需冰浴保存。
5. 请勿把JC-1染色缓冲液（5×）全部配制成JC-1染色缓冲液（1×），本试剂盒使用过程中需直接使用JC-1染色缓冲液（5×）。
6. 如果发现JC-1染色缓冲液（5×）中有沉淀，必须全部溶解后才能使用，为促进溶解可以在37℃加热。
7. CCCP为线粒体电子传递链抑制剂，有毒，请注意小心防护。
8. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product composition
Product name | Package |
JC-1(200×) | 100μL/tube, 5 tubes in total |
Ultra-pure water | 90mL |
JC-1 staining buffer (5×) | 80mL |
CCCP(10mM) | 20μL |
Product Introduction
Mitochondrial Membrane Potential Detection Kit (JC-1) is a kit that uses JC-1 as a fluorescent probe to quickly and sensitively detect changes in cell, tissue or purified mitochondrial membrane potential. It can be used for early apoptosis .
JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential △Ψm. Can detect cell, tissue or purified mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of the mitochondria to form a polymer, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot accumulate in the matrix of the mitochondria, at this time JC- 1 is a monomer, which can produce green fluorescence. In this way, it is very convenient to detect the change of mitochondrial membrane potential through the change of fluorescence color. The relative ratio of red and green fluorescence is commonly used to measure the ratio of mitochondrial depolarization.
The decrease of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. The decrease of cell membrane potential can be easily detected by the transition of JC-1 from red fluorescence to green fluorescence, and the transition from red fluorescence to green fluorescence of JC-1 can also be used as an early detection indicator of cell apoptosis.
The maximum excitation wavelength of JC-1 monomer is 515nm and the maximum emission wavelength is 529nm; the maximum excitation wavelength of JC-1 polymer is 585nm and the maximum emission wavelength is 590nm. For actual observation, use the conventional settings for observing red fluorescence and green fluorescence.
This kit provides CCCP as a positive control for inducing the decrease of mitochondrial membrane potential.
For the samples in the six-well plate, the kit can detect a total of 100 samples; for the samples in the 12-well plate, the kit can detect a total of 200 samples.
Instructions for use
1. Preparation of JC-1 dyeing working solution
The amount of JC-1 staining working solution required for each well of a six-well plate is 1mL, and the amount of JC-1 staining working solution for other culture vessels can be deduced by analogy: 0.5mL JC-1 for every 500,000 to 1 million cells of cell suspension Dyeing working solution. Take an appropriate amount of JC-1 (200×) and dilute JC-1 at the ratio of adding 8mL ultrapure water to every 50μL of JC-1 (200×). Shake vigorously to dissolve and mix JC-1 thoroughly. Then add 2mL JC-1 staining buffer (5×), and mix well to become the JC-1 staining working solution.
2. Setting of positive control
Add the CCCP (10mM) provided in the kit to the cell culture medium at a ratio of 1:1000, dilute to 10μM, and treat the cells for 20 minutes. Then load JC-1 according to the following method to detect the mitochondrial membrane potential. For most cells, the membrane potential of the mitochondria will be completely lost after 20 minutes of treatment with 10μM CCCP. After staining with JC-1, it should show green fluorescence; while normal cells should show red fluorescence after staining with JC-1. For specific cells, the concentration and duration of action of CCCP may be different, so you need to refer to relevant literature to determine.
3. For suspension cells
Take 100,000 to 600,000 cells and resuspend them in 0.5 mL of cell culture medium, which can contain serum and phenol red.
Add 0.5mL JC-1 staining working solution, invert several times to mix. Incubate in a cell incubator at 37°C for 20 minutes.
During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) at the ratio of adding 4 mL of distilled water per 1 mL of JC-1 staining buffer (5×), and place in an ice bath.
After incubation at 37°C, centrifuge at 600g 4°C for 3 to 4 minutes to pellet the cells. Discard the supernatant and try not to aspirate the cells.
Wash twice with JC-1 staining buffer (1×): add 1mL JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600g at 4°C for 3-4 minutes, pellet the cells, and discard the supernatant. Then add 1mL JC-1 staining buffer (1×) to resuspend the cells, centrifuge at 600g at 4°C for 3-4 minutes to pellet the cells and discard the supernatant.
Then resuspend it with an appropriate amount of JC-1 staining buffer (1×) and observe it with a fluorescence microscope or a laser confocal microscope. It can also be detected with a fluorescence spectrophotometer or analyzed by a flow cytometer.
4. For adherent cells
Note: For adherent cells, if you want to use a fluorescence spectrophotometer or flow cytometer to detect, you can collect the cells first, and then refer to the detection method of suspended cells after resuspension.
For one well of the six-well plate, aspirate the culture medium, wash the cells once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1 mL of cell culture medium. The cell culture fluid may contain serum and phenol red.
Add 1mL JC-1 dyeing working solution and mix well. Incubate in a cell incubator at 37°C for 20 minutes.
During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) at the ratio of adding 4 mL of distilled water per 1 mL of JC-1 staining buffer (5×), and place it in an ice bath.
After incubation at 37°C, aspirate the supernatant and wash twice with JC-1 staining buffer (1×).
Add 2 mL of cell culture medium, which can contain serum and phenol red.
Observe under fluorescence microscope or laser confocal microscope.
5. For purified mitochondria
Dilute the prepared JC-1 staining working solution with JC-1 staining buffer (1×) 5 times.
Add 0.1 mL of purified mitochondria with a total protein content of 10-100 μg to 0.9 mL of the 5-fold diluted JC-1 staining working solution.
Detect with a fluorescence spectrophotometer or a fluorescence microplate reader: directly use a fluorescence spectrophotometer to perform a time scan after mixing. The excitation wavelength is 485nm and the emission wavelength is 590nm. If you use a fluorescence microplate reader and the excitation wavelength cannot be set to 485nm, you can set the excitation wavelength within the range of 475~520nm. In addition, you can also refer to the wavelength setting in step 6 below for fluorescence detection.
Observe with a fluorescence microscope or a laser confocal microscope: the method is the same as step 6 below.
6. Fluorescence observation and result analysis
When detecting JC-1 monomer, you can set the excitation light to 490nm and the emission light to 530nm; when detecting JC-1 polymer, you can set the excitation light to 525nm and the emission light to 590nm. Note: It is not necessary to set the excitation light and emission light to the maximum excitation wavelength and maximum emission wavelength when measuring fluorescence here. If you use a fluorescence microscope to observe, you can refer to other green fluorescence settings when detecting JC-1 monomer, such as the setting when observing GFP or FITC; when detecting JC-1 polymer, you can refer to other red fluorescence, such as propidium iodide. Setting when pyridine or Cy3. The appearance of green fluorescence indicates that the mitochondrial membrane potential has decreased, and the cell is likely to be in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the state of the cells is relatively normal.
Storage Conditions
Store at -20°C and avoid light, try to avoid repeated freezing and thawing. The validity period is one year.
Ultrapure water and JC-1 staining buffer (5×) can also be stored at 4°C.
Precautions
1. JC-1 (200×) will solidify and stick to the bottom, wall or cap of the centrifuge tube at a lower temperature such as 4℃ and ice bath. It can be incubated in a water bath at 20～25℃ for a while until it is completely melted. After use.
2. JC-1 (200×) must be fully dissolved and mixed with the ultrapure water provided in the kit before adding JC-1 staining buffer (5×). Do not prepare JC-1 staining buffer (1×) before adding JC-1 (200×). In this way, JC-1 will be difficult to fully dissolve, which will seriously affect subsequent detection.
3. When washing with JC-1 staining buffer (1×) after loading JC-1, keep JC-1 staining buffer (1×) at about 4°C. The washing effect is better at this time.
4. After the JC-1 probe is loaded and washed, try to complete the follow-up test within 30 minutes. It needs to be stored in an ice bath before testing.
5. Do not mix all JC-1 staining buffer (5×) into JC-1 staining buffer (1×). You need to use JC-1 staining buffer (5×) directly during the use of this kit.
6. If it is found that there is precipitation in JC-1 staining buffer (5×), it must be completely dissolved before use. In order to promote the dissolution, it can be heated at 37°C.
7. CCCP is an inhibitor of the mitochondrial electron transport chain. It is toxic. Please take care to protect it.
8. For your safety and health, please wear lab coats and disposable gloves.
规格
英文名称
Mitochondrial Membrane Potential Detection Kit (JC-1)
储存与运输
储存条件
避光,-20°C储存,干燥
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
技术文档和文章
此产品的引用文献
溶液计算器
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