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线粒体膜电位检测试剂盒 (Rhodamine 123)
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为什么选择此级别
BioReagent,无菌,用于细胞培养 级 ，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。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
Rhodamine 123（罗丹明123，Rh 123）是一种常用的阳离子绿色荧光染料，可以透过细胞膜从而在活细胞线粒体内聚集，并发出黄绿色荧光，可广泛用于检测线粒体膜电位，并且对细胞没有任何毒性。
检测线粒体膜电位的核心原理依赖于线粒体跨膜电位（ΔΨm）的静电作用主动穿透并富集于基质：在健康细胞中，高膜电位（-150~180mV）使染料选择性聚集于线粒体内，发出黄绿色荧光（强度与膜电位正相关，激发/发射波长为507nm/529nm）；当细胞发生凋亡或坏死时，膜电位去极化导致染料释放，荧光显著减弱。
| M1505999 | Components | 100-1000T | Storage |
| M1505999A | Rhodamine 123 (1000X） | 100 μL | -20℃. Store in the dark |
| M1505999B | 染色缓存液 | 100 mL | -20℃ |
| M1505999C | CCCP（10mM） | 20 μL | -20℃ |
自备试剂及耗材：吸头/离心管。
实验步骤：
1.Rhodamine123染色工作液的配制：
a.6孔板每孔所需 Rhodamine 123染色工作液的量为1ml，其它培养器皿的 Rhodamine 123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 Rhodamine 123染色工作液。
b.取适量 Rhodamine 123 (1000X)，按照每1μl Rhodamine 123 (1000X) 加入1ml 检测缓冲液的比例稀释 Rhodamine 123，混匀后即为 Rhodamine 123 染色工作液。
注1：配制 Rhodamine 123 染色工作液时注意避光，且须现配现用，不能长期保存。
注2：本试剂盒中提供的检测缓冲液含有Ca²⁺，能够在一段时间内维持细胞的正常状态，并给细胞提供一定的营养，效果比PBS或HBSS更好；检测缓冲液也可用细胞培养液代替，但培养液中不能含有血清，否则会影响 Rhodamine 123 的染色效果。
注3：染色工作液中 Rhodamine 123 的最终浓度需根据不同细胞系和实验体系通过预实验进行优化。Rhodamine 123 的推荐工作浓度为1X，可以在0.1X-5X范围内摸索最佳工作浓度。
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。随后按照下述方法加入 Rhodamine 123 染色工作液，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，Rhodamine 123 染色后观察应呈弱黄绿色或几乎无荧光；而正常的细胞经 Rhodamine 123 染色后应显示明亮的黄绿色荧光。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或自行摸索确定。
3.对于悬浮细胞：
a.细胞按照实验设计进行一定处理后，计数。取适量细胞600×g室温离心5min，弃上清，加入适当体积的 Rhodamine 123 染色工作液重悬细胞，使细胞密度约为 1×106/ml。
b.细胞培养箱中37°C孵育20-60min，不同的细胞最佳孵育时间不同。以20min作为初始孵育时间，对孵育时间进行适当优化以得到最佳的效果。
c.37°C孵育结束后，600×g室温离心5min，沉淀细胞。吸除上清，注意尽量不要触及细胞。
d.用37°C预热的细胞培养液洗涤2次：加入1ml37°C预热的细胞培养液重悬细胞，离心5分钟，沉淀细胞，弃上清；重复一次。
e.再用适量细胞培养液重悬后，用荧光显微镜或激光共聚焦显微镜观察，也可以用荧光酶标仪或流式细胞仪分析。
4.对于贴壁细胞：
注意：对于贴壁细胞，如果希望采用荧光酶标仪或流式细胞仪检测，可以先收集细胞，重悬后参考悬浮细胞的检测方法。荧光酶标仪如果能采用底读的方式进行荧光检测，也可以使用96孔板等进行贴壁培养检测的。
a.对于6孔板的一个孔的细胞，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。
b.加入1ml Rhodamine 123 染色工作液。细胞培养箱中37°C孵育20-60min，不同的细胞最佳孵育时间不同。以20min作为初始孵育时间，对孵育时间进行适当优化以得到最佳的效果。
c.37°C孵育结束后，吸除上清，用预热的细胞培养液洗涤2次。
d.加入2ml预热的细胞培养液，培养液中可以含有血清和酚红。
e.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：
a.准备好 Rhodamine 123 染色工作液。
b.0.9ml Rhodamine 123 染色工作液中加入0.1ml总蛋白量为10-100μg纯化的线粒体。
c.用荧光酶标仪或荧光分光光度计检测：混匀后直接用荧光分光光度计进行时间扫描(timescan)，激发波长为507nm，发射波长为529nm。如果使用荧光酶标仪，可在软件中把检测对象设置为FITC，即可以检测 Rhodamine 123。另外，也可以参考下面步骤6中的波长设置进行荧光检测。
d.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：
Rhodamine 123 的最大激发波长为507nm，最大发射波长为529nm。如使用荧光显微镜观察，可以参考观察FITC等其它绿色荧光时的设置。黄绿色荧光变弱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。通过对比实验组与阴性对照组测量的Relative Fluorescence Units (RFU)，可以得出药物处理后线粒体内Rhodamine123探针荧光强度的变化。此处的阴性对照为仅含检测缓冲液的未经染色的细胞样品。
注：流式检测应获得两个相对独立的细胞群：明亮绿色荧光的正常细胞群和弱绿色荧光的凋亡或坏死细胞群。
注意事项：
1.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。
2.本试剂盒仅限于用于存活的细胞或组织的检测，不可用于固定或冻存的细胞或组织样品的检测。
3.检测缓冲液经过过滤除菌处理，在使用时须注意避免微生物污染，否则很可能严重影响染色效果。如果检测缓冲液发生浑浊等明显的微生物污染，就不能继续使用。
4.CCCP为线粒体电子传递链抑制剂，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。
5.荧光酶标仪检测时须使用适合荧光检测的黑板或白板，建议使用96孔黑板。
6.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。
7.为了您的安全和健康，请穿实验服并戴一次性手套操作。
Rhodamine 123 (Rhodamine 123, Rh 123) is a commonly used cationic green fluorescent dye that can permeate cell membranes, accumulate within mitochondria of live cells, and emit yellowish-green fluorescence. It is widely used for detecting mitochondrial membrane potential and is non-toxic to cells.
The core principle of detecting mitochondrial membrane potential relies on the electrostatic action of the mitochondrial transmembrane potential (ΔΨm) to actively penetrate and enrich the dye in the matrix: In healthy cells, a high membrane potential (-150~180mV) causes the dye to selectively accumulate within mitochondria, emitting yellowish-green fluorescence (the intensity is positively correlated with the membrane potential, with excitation/emission wavelengths at 507nm/529nm). When cells undergo apoptosis or necrosis, membrane potential depolarization leads to dye release, resulting in a significant decrease in fluorescence.
| M1505999 | Components | 100-1000T | Storage |
| M1505999A | Rhodamine 123 (1000X） | 100 μL | -20℃. Store in the dark |
| M1505999B | Staining Buffer Solution | 100 mL | -20℃ |
| M1505999C | CCCP（10mM） | 20 μL | -20℃ |
Self-Prepared Reagents and Consumables: Pipette Tips/Centrifuge Tubes.
Experimental Procedures:
1. Preparation of Rhodamine 123 Staining Working Solution:
a. The volume of Rhodamine 123 Staining Working Solution required per well of a 6-well plate is 1 mL; the dosage of Rhodamine 123 Staining Working Solution for other culture vessels can be deduced by analogy. For cell suspensions, 0.5 mL of Rhodamine 123 Staining Working Solution is needed for every 500,000–1,000,000 cells.
b. Take an appropriate amount of Rhodamine 123 (1000X), and dilute it by adding 1 μL of Rhodamine 123 (1000X) to 1 mL of Assay Buffer. After mixing well, the resulting solution is the Rhodamine 123 Staining Working Solution.
Note 1: When preparing the Rhodamine 123 Staining Working Solution, avoid light exposure. The solution must be prepared fresh and used immediately; long-term storage is not allowed.
Note 2: The Assay Buffer provided in this kit contains Ca²⁺, which can maintain the normal state of cells for a certain period and provide the cells with a certain amount of nutrients. Its effect is better than that of PBS or HBSS. The Assay Buffer can also be replaced with cell culture medium, but the medium must not contain serum—otherwise, the staining effect of Rhodamine 123 will be affected.
Note 3: The final concentration of Rhodamine 123 in the Staining Working Solution needs to be optimized through preliminary experiments according to different cell lines and experimental systems. The recommended working concentration of Rhodamine 123 is 1X, and the optimal working concentration can be explored within the range of 0.1X–5X.
2. Setup of Positive Control:
Add the CCCP (10 mM) provided in the kit to the cell culture medium at a recommended dilution ratio of 1:1000 to dilute it to 10 μM, and treat the cells with this solution for 20 minutes. Then add the Rhodamine 123 Staining Working Solution according to the method described below to detect the mitochondrial membrane potential. For most cells, the mitochondrial membrane potential usually completely dissipates after treatment with 10 μM CCCP for 20 minutes, and weak yellow-green fluorescence or almost no fluorescence should be observed after Rhodamine 123 staining. In contrast, normal cells should exhibit bright yellow-green fluorescence after staining with Rhodamine 123. For specific cell types, the effective concentration and treatment time of CCCP may vary. It is necessary to refer to relevant literature or conduct independent optimization experiments to determine the appropriate conditions.
3. For Suspension Cells:
a. After treating the cells according to the experimental design, count them. Take an appropriate amount of cells and centrifuge at 600×g for 5 minutes at room temperature. Discard the supernatant, then add an appropriate volume of Rhodamine 123 Staining Working Solution to resuspend the cells, adjusting the cell density to approximately 1×10⁶ cells/mL.
b. Incubate the cells at 37°C in a cell incubator for 20–60 minutes. The optimal incubation time varies among different cell types. Use 20 minutes as the initial incubation time, and optimize the incubation time appropriately to achieve the best results.
c. After incubation at 37°C, centrifuge the cells at 600×g for 5 minutes at room temperature to pellet the cells. Aspirate and remove the supernatant, taking care to avoid touching the cell pellet as much as possible.
d. Wash the cells twice with cell culture medium pre-warmed to 37°C: Add 1 mL of 37°C pre-warmed cell culture medium to resuspend the cells, centrifuge for 5 minutes to pellet the cells, and discard the supernatant; repeat this step once.
e. Resuspend the cells again in an appropriate amount of cell culture medium, then observe using a fluorescence microscope or laser confocal microscope. Alternatively, analyze using a fluorescence microplate reader or flow cytometer.
4. For Adherent Cells:
Note: For adherent cells, if detection via a fluorescence microplate reader or flow cytometer is desired, collect the cells first, resuspend them, and then refer to the detection method for suspension cells. If the fluorescence microplate reader supports bottom-reading for fluorescence detection, adherent cells can also be cultured and detected in a 96-well plate or similar vessels.
a. For cells in one well of a 6-well plate, aspirate and remove the culture medium. If necessary according to the specific experiment, wash the cells once with PBS or another appropriate solution.
b. Add 1 mL of Rhodamine 123 Staining Working Solution. Incubate the cells at 37°C in a cell incubator for 20–60 minutes. The optimal incubation time varies among different cell types. Use 20 minutes as the initial incubation time, and optimize the incubation time appropriately to achieve the best results.
c. After incubation at 37°C, aspirate and remove the supernatant, then wash the cells twice with pre-warmed cell culture medium.
d. Add 2 mL of pre-warmed cell culture medium; the medium may contain serum and phenol red.
e. Observe the cells under a fluorescence microscope or laser confocal microscope.
5. For Purified Mitochondria:
a. Prepare the Rhodamine 123 Staining Working Solution.
b. Add 0.1 mL of purified mitochondria with a total protein content of 10–100 μg to 0.9 mL of Rhodamine 123 Staining Working Solution.
c. Detection with a fluorescence microplate reader or fluorescence spectrophotometer: After mixing well, directly perform a time scan using a fluorescence spectrophotometer with an excitation wavelength of 507 nm and an emission wavelength of 529 nm. If using a fluorescence microplate reader, set the detection target to FITC in the software to detect Rhodamine 123. Alternatively, refer to the wavelength settings in Step 6 below for fluorescence detection.
d. Observation with a fluorescence microscope or laser confocal microscope: Follow the method described in Step 6 below.
6. Fluorescence Observation and Result Analysis:
The maximum excitation wavelength of Rhodamine 123 is 507 nm, and its maximum emission wavelength is 529 nm. When observing with a fluorescence microscope, the settings used for other green fluorescent substances (such as FITC) can be referenced. A decrease in yellow-green fluorescence indicates a reduction in mitochondrial membrane potential, and the cells are likely in the early stage of apoptosis. By comparing the Relative Fluorescence Units (RFU) measured between the experimental group and the negative control group, the change in the fluorescence intensity of the Rhodamine 123 probe in mitochondria after drug treatment can be determined. The negative control here refers to unstained cell samples containing only the assay buffer.
Note: For flow cytometry detection, two relatively distinct cell populations should be obtained: a population of normal cells with bright green fluorescence, and a population of apoptotic or necrotic cells with weak green fluorescence.
Precautions:
1. All fluorescent dyes have the issue of photobleaching. Please take precautions to avoid light exposure as much as possible to slow down fluorescent photobleaching.
2. This kit is only intended for the detection of viable cells or tissues, and must not be used for the detection of fixed or cryopreserved cell or tissue samples.
3. The assay buffer has been filter-sterilized. During use, care must be taken to avoid microbial contamination, as this is likely to seriously affect the staining effect. If the assay buffer becomes turbid or shows other obvious signs of microbial contamination, it must not be used further.
4. CCCP is an inhibitor of the mitochondrial electron transport chain and is harmful to the human body. Please handle it with caution during operation and take effective protective measures to avoid direct contact with the body or inhalation.
5. When detecting with a fluorescence microplate reader, black plates or white plates suitable for fluorescence detection must be used; the use of 96-well black plates is recommended.
6. This product is solely for scientific research use by professional personnel. It must not be used for clinical diagnosis or treatment, nor for food or pharmaceutical purposes, and must not be stored in ordinary residential premises.
7. For your safety and health, please wear a lab coat and disposable gloves during operation.
规格
英文别名
Mitochondrial Membrane Potential Detection Kit
规格或纯度
BioReagent,用于细胞培养,无菌
英文名称
Mitochondrial Membrane Potential Assay Kit (Rhodamine 123)
应用
Live/Dead Cell Assay, 细胞凋亡, 细胞分析, 细胞染色
敏感性
Light-sensitive
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131010 |
分析证书 | M1505999 |
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