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.
