Dihydrorhodamine 123 (DHR 123)
(Synonyms: 二氢罗丹明123; DHR 123) 目录号 : GC30581
Dihydrorhodamine 123 (DHR 123)是一种荧光探针，主要用于活性氧(ROS)种类的检测。激发波长λex=485 nm，发射波长λem=535 nm。
Cas No.:109244-58-8
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1.溶液配制
(1)制备储存液：将低温保存的固体平衡至室温，使用DMSO溶解Dihydrorhodamine 123，配置浓度为5mM的储存液。
注意:未使用的储存液分装后在-20℃或-80°C避光保存，避免反复冻融。
(2)制备工作液：正式实验前，用合适的缓冲液(如:无血清培养基或PBS)稀释储存液，到所需的工作浓度。
注意：最佳的工作浓度请根据实际情况调整或参阅文献自行设置梯度浓度进行摸索。工作液必须现配现用
2.用DHR123检测细胞内ROS步骤[1](来自文献,仅做参考)
(1) 细胞处理：用水清洗细胞以去除任何污染物或碎片。
(2) DHR123孵育：将细胞重悬于含有DHR123（15 μg/ml，约43μM）的PBS中。在30°C 下振荡孵育 90min。
(3)洗涤细胞：用PBS缓冲液清洗细胞沉淀两次，去除多余的 DHR123。
(4)荧光检测：用流式细胞仪测量 10,000个细胞的荧光。将激发和发射波长分别设置为 485 nm 和 535 nm，检测DHR123与细胞内ROS反应发出的荧光。使用 FlowJo 7.6.5 软件分析样品中的荧光分布。
3. 用DHR123检测细胞内ROS步骤[2](来自文献,仅做参考)
(1) 细胞处理：根据实验要求处理细胞。
(2) DHR123孵育：在HBSS缓冲盐溶液中制备含有2.5 μM DHR123的溶液。将细胞与 DHR123 溶液在 37°C 下孵育30 min。
(3)洗涤细胞：用HBSS洗涤细胞两次，以去除多余的 DHR123。
(4)裂解细胞：使用 RIPA 缓冲液裂解细胞以释放细胞内转化的Rh123+。
(5)荧光检测：将激发和发射波长分别设置为 485 nm 和 535 nm，以检测 Rh123+荧光。将荧光强度读数记录为指示细胞内ROS水平的相对单位。
注意：该实验方案为使用Dihydrorhodamine 123检测细胞内活性氧 (ROS) 和随后的荧光测量提供指导。可以根据具体实验要求进行调整。
Dihydrorhodamine 123 (DHR 123) is a non-fluorescent precursor that transforms into the fluorescent compound rhodamine 123 (Rh123+) upon oxidation. This transformation process makes DHR 123 a useful tool for detecting reactive oxygen species (ROS) within cells. Dihydrorhodamine 123 can cross the cell membrane, and once inside the cell, it is oxidized by ROS into Rh123+, producing green fluorescence that accumulates in the mitochondrial matrix. The excitation wavelength of Rh123+ is 485 nm, and its emission wavelength is 535 nm[1]. Dihydrorhodamine 123 can also be used as an indicator for the formation of intracellular peroxynitrite[2].
References:
[1] Wing-Kee Lee, Stephanie Probst, Bettina Scharner, et al. Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line[J] Archives of Toxicology. 30 January 2024.
[2] Crow, J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species Nitric Oxide1(2)145-157(1997).
Dihydrorhodamine 123 (DHR 123)是一种非荧光前体，它在氧化时转化为荧光化合物rhodamine 123(Rh123+ )。这种转换过程使得DHR 123成为检测细胞内活性氧种类（ROS）的有用工具。Dihydrorhodamine 123可以穿过细胞膜，进入细胞内部会被活性氧氧化成Rh123+，产生绿色荧光，在线粒体基质中积累。Rh123+激发波长为485 nm，发射波长为535 nm[1]。Dihydrorhodamine 123也可以用作细胞内过氧亚硝酸盐形成的指示剂[2]。
| Cas No. | 109244-58-8 | SDF | |
| 别名 | 二氢罗丹明123; DHR 123 | ||
| Canonical SMILES | O=C(OC)C1=CC=CC=C1C2C3=C(OC4=C2C=CC(N)=C4)C=C(N)C=C3 | ||
| 分子式 | C21H18N2O3 | 分子量 | 346.38 |
| 溶解度 | DMSO: 100 mg/mL (288.70 mM) | 储存条件 | Store at -20°C,protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 2.887 mL | 14.435 mL | 28.87 mL |
| 5 mM | 0.5774 mL | 2.887 mL | 5.774 mL |
| 10 mM | 0.2887 mL | 1.4435 mL | 2.887 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Ameliorating effects of traditional Chinese medicine preparation, Chinese materia medica and active compounds on ischemia/reperfusion-induced cerebral microcirculatory disturbances and neuron damage
Ischemic stroke and ischemia/reperfusion (I/R) injury induced by thrombolytic therapy are conditions with high mortality and serious long-term physical and cognitive disabilities. They have a major impact on global public health. These disorders are associated with multiple insults to the cerebral microcirculation, including reactive oxygen species (ROS) overproduction, leukocyte adhesion and infiltration, brain blood barrier (BBB) disruption, and capillary hypoperfusion, ultimately resulting in tissue edema, hemorrhage, brain injury and delayed neuron damage. Traditional Chinese medicine (TCM) has been used in China, Korea, Japan and other Asian countries for treatment of a wide range of diseases. In China, the usage of compound TCM preparation to treat cerebrovascular diseases dates back to the Han Dynasty. Even thousands of years earlier, the medical formulary recorded many classical prescriptions for treating cerebral I/R-related diseases. This review summarizes current information and underlying mechanisms regarding the ameliorating effects of compound TCM preparation, Chinese materia medica, and active components on I/R-induced cerebral microcirculatory disturbances, brain injury and neuron damage.
Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells
Dihydrorhodamine 123 (DHR 123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydrorhodamine 6G (DHR 6G) were evaluated as probes for detecting cellular hydrogen peroxide levels in SPC-A-1 lung adenocarcinoma cells. Imaging techniques and fluorescence-activated cell scan were used in the study of the probe responses. Obvious green fluorescence was established after a 25-min exposure. After staining with MitoTracker Orange CM-H2TMRos (a probe for mitochondria) and the abovementioned probes simultaneously, only the DHR 123 and DHR 6G groups exhibited legible green fluorescence in the mitochondrial regions. Furthermore, the DHR 6G group exhibited weaker fluorescence intensity. When 100 microM H2O2 was added to SPC-A-1 cells loaded with these probes, the intracellular fluorescence increased rapidly and significantly. Our results suggest that DHR 123 is superior for the instantaneous detection of cellular hydrogen peroxide in SPC-A-1 cells.
Reliability of acute illness dihydrorhodamine-123 testing for chronic granulomatous disease
Background: Dihydrorhodamine (DHR) flow cytometric analysis is used to evaluate granulocyte oxidative bursts and is the test of choice for the diagnosis of chronic granulomatous disease (CGD). We present the clinical and DHR test profiles of five subjects assessed during and after acute illness.
Methods: This was a retrospective report of the findings of five out of a total of one hundred and seventeen patients, whose blood was sent to the laboratory for dihydrorhodamine-123 flow cytometry testing between January 2005 and December 2010. Using whole blood technique and stimulation using phorbol myristate acetate, the results of DHR were expressed as stimulation index and coefficient of variation of histograms of stimulated cells and compared with healthy controls. DHR tests were repeated when the patients had recovered and were clinically well.
Results: These five patients showed abnormal DHR test results during their acute illness, with a stimulation index (SI) lower (p = 0.009) and coefficient of variation (CV) higher (p = 0.009) than controls. The DHR profiles repeated when patients had recovered showed normalization of tests with no significant difference for SI (p = 0.602) and CV (p = 0.917) compared to controls. Wilcoxon Signed Rank tests showed a significant improvement in SI (p = 0.043) and CV (p = 0.043) upon recovery. On follow up, all five patients were well, with no further severe or atypical infections.
Conclusions: DHR may be transiently abnormal during acute illness, and may therefore not be reliable when assessed during an acute illness. If these subjects had CGD, it would be of a hypomorphic variant that has not previously been described.
Dihydrorhodamine 123: a fluorescent probe for superoxide generation?
Imaging techniques, such as confocal microscopy and fluorescent activated cells scan are facilitating the study of responses at the single-cell level. Superoxide is reported to oxidise the non-fluorescent dihydrorhodamine 123 (DHR) to rhodamine 123. The generation of rhodamine 123 by human neutrophils, stimulated by the phorbol ester phorbol 12-myristate 13-acetate was inhibited slowly by diphenylene iodonium and rapidly by azide, but not by superoxide dismutase. In the absence of enzymes H2O2 (but not O2-.) oxidised DHR slowly but the rate was greatly enhanced by peroxidases. The rhodamine 123 generated by phorbol-ester-stimulated neutrophils was observed to be located within the cell despite the fact that neutrophils failed to accumulate external rhodamine 123. This stimulated rise in cellular fluorescence was eliminated by excess extracellular catalase. It appears that H2O2, released on the outside, crosses the plasma membrane where oxidation of DHR is catalysed by cellular peroxidases. Since in a mixed population DHR failed to distinguish between O2-.-producing and non-producing HL60 cells it is not a suitable probe for single-cell observations. We conclude that DHR oxidation reports only the presence of H2O2 and intracellular peroxidases, and not the generation of O2-. by any one cell. Only peroxidase-containing cells fluoresce.
Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro
Reactive oxygen species (ROS) are formed in biological systems by partial reduction of molecular oxygen. The essential role of ROS in maintaining physiological health may be corrupted into oxidative stress by their overproduction or the exhaustion of antioxidant mechanisms. Many studies covering a broad range of methodologies have investigated ROS production and their toxic mechanisms of action. Of these methodologies, fluorometry has been among the preferred techniques. Three frequently used fluorescent probes for in vitro studies are 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA), Dihydrorhodamine 123 (DHR 123) and Dihydroethidium (DHE). Apart from the unavoidable limitations of auto-oxidation, photo-oxidation and photo-conversion, there are also concerns relating to protocol modification for the improved monitoring of ROS. This paper aims to highlight such contributing factors, including cell culture conditions and the characteristics of individual fluorescent probes in the utilization of these selected probes in in vitro systems.