Mitochondrial Permeability Transition Pore (MPTP) Assay Kit
Low toxicity does not impair cellular functions; precise analysis of pore opening degree.
One-click copy of information
Instruction manualPrice:
¥ 1000
Item Number:
100-118
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Specification
- 50-500T
隐藏域元素占位
- Product Description
- Composition
- Precautions
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Product Storage:
Store at −20°C; stable for one year. Please protect Calcein AM (1000×) and Ionomycin (200×) from light.
Product Overview:
The Mitochondrial Permeability Transition Pore Assay Kit (MPTP Assay Kit) enables the assessment of mitochondrial permeability transition pore opening using the membrane-permeable fluorescent probe Calcein AM. It is widely employed in research on cell death pathways, including apoptosis and necrosis.
This kit uses the nonpolar dye Calcein AM to fluorescently stain live cells. After entering the cell, Calcein AM accumulates in the cytoplasm, including within mitochondria. Intracellular esterases hydrolyze Calcein AM, converting it into the membrane‑impermeable, polar fluorescent dye calcein (also known as Fluorexon), which emits intense green fluorescence. Calcein is a metal‑complexing indicator; when bound to metal ions such as Co²⁺, its fluorescence is quenched. Under normal conditions, the mitochondrial permeability transition pore (MPTP) remains closed, preventing CoCl₂ from entering the mitochondria. Consequently, following Calcein AM staining and subsequent treatment with CoCl₂, fluorescence in the cytoplasm is quenched, leaving only the mitochondria displaying green fluorescence. If, at this point, the calcium ionophore ionomycin is used to induce extracellular Ca²⁺ uptake into both the cytoplasm and mitochondria, the MPTP may open to varying degrees. Under these circumstances, some of the calcein stored in the mitochondria will be released into the cytosol, where it binds to cobalt ions and loses its fluorescence, while the cobalt ions…
The probe enters the mitochondria, quenching all or part of the green fluorescence of calcein within the organelles, ultimately leading to a reduction or disappearance of mitochondrial green fluorescence. By monitoring the intensity of green fluorescence in the mitochondria, one can assess the degree of MPTP opening: stronger fluorescence indicates lower MPTP opening, while weaker fluorescence indicates higher MPTP opening.
The hydrolysis product of Calcein AM, Calcein, exhibits a maximum excitation wavelength of 494 nm and a maximum emission wavelength of 517 nm.
Calcein AM exhibits extremely low cytotoxicity and does not inhibit most cellular functions, such as proliferation and chemotaxis; therefore, compared with similar reagents like CFDA‑SE, it is a more suitable fluorescent probe for live-cell staining.
Instructions for Use
I. Preparation of the reaction solution:
1. Prepare Calcein AM staining solution (using flow cytometry as an example; 10 mL of Calcein AM staining solution is required for 10 samples):
Add 10 μL of Calcein AM (1000×) to 10 mL of assay buffer and mix thoroughly.
Please note:
a) Calcein AM staining solution should be prepared fresh and used immediately; do not store it for extended periods.
b) The assay buffer provided with this kit contains Ca²⁺, which helps maintain cells in a normal state for a limited period and supplies adequate nutrients. This buffer can be replaced with cell culture medium; however, serum‑containing media should be avoided, as serum reduces the efficiency of Calcein AM uptake into cells.
c) The final concentration of Calcein AM in the staining solution should be adjusted according to the specific experimental conditions. It can be varied within the range of 0.1× to 5×.
2. Prepare the fluorescence quenching working solution:
Add CoCl2 (200×) to the Calcein AM staining solution at a ratio of 1:200, thereby diluting the CoCl2 200-fold to a final concentration of 1×. For example, add 50 μL of CoCl2 (200×) to every 10 mL of Calcein AM staining solution and mix thoroughly.
Note: The recommended final concentration of CoCl₂ is 1×; this typically yields good quenching efficiency. The final concentration of CoCl₂ can be optimized based on experimental conditions and adjusted within the range of 0.1× to 1×.
3. Preparation of Ionomycin Control:
Add Ionomycin (200×) to the fluorescence quenching working solution at a 1:200 ratio, thereby diluting Ionomycin 200-fold to a final concentration of 1×. For example, add 50 μL of Ionomycin (200×) to every 10 mL of the fluorescence quenching working solution and mix thoroughly. Depending on the experimental conditions, the final concentration of Ionomycin can be adjusted within the range of 0.4× to 6×.
4. Details of solution preparation are provided in the table below. The table uses the preparation of 10 mL of each solution as an example:
solution Solvent Reagent Concentrated solution concentration Final concentration used Volume Calcein AM staining solution (A) Detection buffer Calcein AM 1000× 1 × 10 μL Fluorescence Quenching Working Solution (B) A CoCl2 200× 1 × 50 μL Ionomycin control (C) B Ionomycin 200× 1 × 50 μL II. Fluorescence Microscopy of Adherent Cells:
1. Seed the cells in an appropriate culture dish. Remove the culture medium and wash the cells 1–2 times with PBS.
2. Add appropriate volumes of Calcein AM staining solution, fluorescence quenching working solution, or Ionomycin control, and gently shake to ensure the dye evenly covers all cells. Typically, 100 μL of liquid is added per well in a 96-well plate, 250 μL per well in a 24-well plate, and for a 12-well plate, the volume per well is…
500 μL; the volume of liquid added to each well of a 6-well plate is 1 mL.
3. Incubate at 37°C in the dark for 30–45 minutes (the optimal incubation time depends on the cell type; please determine the best conditions by testing with your specific cell line).
4. Remove the reaction solution and add fresh, 37°C–prewarmed culture medium to the cells. Incubate in the dark at 37°C for 30 minutes to ensure complete hydrolysis of Calcein AM by intracellular esterases, yielding Calcein with green fluorescence.
5. Remove the culture medium, wash 2–3 times with PBS, and then add the detection buffer before observing under a fluorescence microscope. Note that the entire assay must be performed in the dark.
III. Fluorescence Microscopy of Suspension Cells:
1. Count the cells to be analyzed. Centrifuge an appropriate cell sample at 1,000 × g at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in a suitable volume of Calcein AM staining solution, fluorescence quenching working solution, or Ionomycin control, adjusting the cell density to approximately 1 × 10^6/mL.
2. Incubate at 37°C in the dark for 30–45 minutes (the optimal incubation time depends on the cell type; please determine the best conditions by testing with your specific cell line).
3. After incubation, centrifuge at 1000×g at room temperature for 5 minutes, discard the supernatant, and gently resuspend the cells in fresh culture medium pre-warmed to 37ºC.
4. Repeat step 3 two or more times.
5. Incubate at 37°C in the dark for 30 minutes to ensure that intracellular esterases fully hydrolyze Calcein AM, generating Calcein with green fluorescence.
6. Centrifuge at 1000 × g at room temperature for 5 min, remove most of the culture medium, resuspend the cells, prepare smears, and examine them under a fluorescence microscope. Note that all steps of the assay must be performed in the dark.
4. Flow Cytometry Analysis:
1. Use trypsin to digest adherent cells, collect the cells in an Eppendorf tube, and resuspend them in culture medium; suspension‑type cells can be used directly. Count the cells to be analyzed, then centrifuge an appropriate aliquot at 1,000 × g at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in a suitable volume of assay buffer, Calcein AM staining solution, fluorescence quenching working solution, or Ionomycin control solution to prepare a single‑cell suspension, adjusting the cell density to 1 × 10^6 cells/mL. The volume for each sample should be 1 mL.
Note: Prepare a cell sample containing only the assay buffer to serve as the negative control for flow cytometry analysis.
2. Incubate at 37°C in the dark for 30 min.
3. After incubation, centrifuge at 1,000 × g at room temperature for 5 minutes, discard the supernatant, and collect the cells. Add 1 mL of assay buffer to each sample, gently resuspend, and centrifuge at 1,000 × g at room temperature for 5 minutes to pellet the cells. Note: This step removes excess dye and any reagents that may cause fluorescence quenching.
4. Resuspend the cells in 400 µL of assay buffer. Note: Perform all steps under light‑protected conditions. After staining, keep the sample on ice; it can be analyzed by flow cytometry within 1 hour.
5. When setting up the negative control for flow cytometry, use a cell sample containing only the assay buffer and no staining as the control. The maximum excitation wavelength of Calcein is 494 nm, and the maximum emission wavelength is 517 nm.
Results explanation:
A. Containing only Calcein AM: Both mitochondria and the cytoplasm exhibit green fluorescence, with a strong fluorescence signal;
B. Containing Calcein AM and CoCl2: Only mitochondria exhibit green fluorescence, with moderate fluorescence intensity.
C. Containing Calcein AM, CoCl2, and Ionomycin: Both mitochondrial and cytoplasmic green fluorescence are quenched, resulting in a weak or nearly absent fluorescent signal.
The changes in the green fluorescence signals of B and C indicate that the mitochondrial permeability transition pore has been opened, leading to the release of some calcein from the mitochondria and the influx of cobalt ions into the mitochondria, which in turn causes quenching of calcein’s green fluorescence.
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Product Composition
Item number Component (50–500T)* Specifications 100-118A Detection buffer 100 mL 100-118B Calcein AM (1000×) 50 μL 100-118C CoCl2 (200×) 500 μL 100-118D Ionomycin (200×) 250 μL *For flow cytometry, the assay volume per sample is 1 mL, allowing this kit to perform 50 assays; for microscopic observation, the assay volume per well in a 96-well plate is 100 μL, enabling 500 assays with this kit.
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Precautions
1. This kit contains fluorescent reagents and must be stored away from light.
2. Calcein AM (1000×) should not be subjected to repeated freeze-thaw cycles. If the entire amount cannot be used at once, please aliquot and store accordingly.
3. CoCl2 is corrosive and exhibits teratogenic and carcinogenic toxicity; it is toxic to specific human organs, including the respiratory and reproductive systems. Handle with care and ensure adequate protective measures to prevent direct skin contact or inhalation. Also, take precautions to avoid corrosion of other materials. CoCl2 is toxic or harmful to aquatic organisms; do not discharge it directly into the environment.
4. This product is intended for research use only and shall not be used for clinical diagnosis or treatment, nor for food or pharmaceutical purposes, and must not be stored in ordinary residential premises.
5. During the experiment, please wear a lab coat and use gloves and a mask.
Keywords
GOONIE
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