| Usage | 1. Reagent preparation:
(1) PBS buffer (pH7.4)
(2) or HbSS (hank's balanced salt solution)
2. Consumables preparation
(1) centrifuge tube
(2) suction tip
(3) disposable gloves
3. Dyeing working solution preparation:
(1) according to the number of samples, prepare the staining buffer according to the following ratio. Take 100μ L reagent C used 900μ Dilute with L pure water and mix well to form staining buffer
(2) every 500 mu; L staining buffer, add 5ul Ao staining solution and 10ul PI staining solution, and mix well to form the staining working solution
4. Suspension cell staining
(1) collect sample cells, and the number of cells is within 10x105
(2) wash the cells twice with PBS
(3) use 500 mu; L staining working solution to resuspend the cells
(4) incubate for 10-20 minutes at 4 ℃ in the dark after gently mixing
(5) wash the cells with PBS
(6) the results were detected by fluorescence microscope or flow cytometry
5. In situ staining of adherent cells
(1) wash the cells twice with PBS
(2) add an appropriate volume of staining working solution to the cell culture plate or cell climbing piece
(3) incubate the cells at 37 ℃ for 10-20 minutes, and the optimal culture time is different for different cells. 20 min can be used as the initial incubation time, after which the system can be optimized to obtain uniform labeling results
(4) blot the staining working solution, wash the culture plate or coverslip with medium for 2-3 times, cover all cells with preheated medium each time, and then blot the medium
Result Analysis:
Under the fluorescence microscope, 488nm excitation light was used for microscopic examination:
the DNA of normal cells stained with AO was uniform yellow or yellow green, and the morphology and structure were normal
when cells are apoptotic, chromatin condenses, and nuclei are fragmented into punctate, which are stained in a dense and dense manner with different sizes
Necrotic cells showed strong red fluorescence |
| Description | AO/PI double staining apoptosis detection kit is a commonly used staining method for apoptosis morphology research, which uses AO/PI probe to double stain the nucleus to detect the state of apoptotic cells
Acridine orange (AO) is a fluorescent dye with cell membrane permeability. The dye can penetrate the living cell membrane and stain nuclear DNA and RNA. It is different from the binding amount of DNA and RNA in cells. The complex can emit different colors of fluorescence. Ao emits green fluorescence when it is combined with dsDNA, and red fluorescence when it is combined with ssDNA and RNA. When bound to DNA, it is very similar to fluorescein in spectrum, with an excitation maximum of 502nm and an emission maximum of 525nm (green). When it binds to RNA, the excitation maximum shifts to 460nm (blue) and the emission maximum shifts to 650nm (red)
Under the fluorescence microscope, acridine orange can penetrate the normal cell membrane, making the nucleus green or yellowish green uniform fluorescence; In apoptotic cells, apoptotic bodies are formed due to chromatin pyknosis or fragmentation into fragments of different sizes. Acridine orange causes it to be stained with dense yellow green fluorescence, or yellow green fragment particles; However, the yellow fluorescence of necrotic cells decreased or even disappeared
Propidium iodide is a DNA binding dye. Its excitation and emission wavelengths are 488nm and 630nm, respectively. It produces red fluorescence, but it has no membrane permeability, cannot penetrate the membrane of living cells, and can only dye dead cells. Therefore, under the fluorescence microscope, normal cells cannot be colored, early apoptotic cells show weak red light, late apoptotic cells show enhanced red light, and necrotic cells show strong red fluorescence. Product components:
| Component | Name | Specification | Save | | Component A | AO staining solution | 500ul | 2-8 ℃, protected from light | Component B | PI staining solution | 1000ul | 2-8 ℃, protected from light | Component C | Reagent C | 10ml | 2-8 ℃, protected from light |
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| General Notes | 1. The reagent in the screw cap micro reagent tube should be centrifuged briefly before opening the cap, and the liquid on the inner wall of the cap should be collected to the bottom of the tube to avoid liquid spilling when opening the cap
2. Careful operation is required for cell treatment to avoid artificial damage to cells
3. This staining kit can be used for cells, cell smears, etc. The following is an example of fluorescence microscopy detection of suspension cultured cells. For other methods, please refer to relevant materials
4. Adherent cells can be digested and stained, or directly stained in situ. |
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