AO staining solution (1 mg/ml)
AO staining solution (1 mg/ml)
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Product Specification
| Usage | 1. Collect cells (fix the cells first when using flow cytometry), wash the cells once with PBS, count the cells, and adjust the cell concentration to 106/ml. 2. Take an appropriate amount of cell suspension and add Acridine Orange Stain (1 mg/ml) to make the final AO concentration between 8.5 and 17 μg/ml. Mix gently. 3. Stain at room temperature in the dark for 15 minutes. Place the cells onto a glass slide and cover slip with a glass cover slip or analyze on a flow cytometer. 4. Observe under a fluorescence microscope (excitation filter wavelength 488nm, blocking filter wavelength 515nm), count, and photograph. Staining Results: Normal cells: Cells are uniformly stained with a yellow-green fluorescence. Apoptotic cells: Chromatin is condensed, and the nucleus is fragmented into punctate shapes, which are stained as dense, intensely stained green granules of varying sizes. |
| Description | Acridine Orange is a tricyclic heteroaromatic dye that can label DNA and RNA, and is a metachromatic fluorescent dye. This dye is membrane-permeable and can penetrate cell membranes to stain nuclear DNA and RNA. Therefore, AO is often used for detecting intracellular DNA and RNA. AO binds to nucleic acids in two main ways: 1. Intercalation, where AO intercalates between base pairs in double-stranded nucleic acids. This binding mode primarily occurs with DNA, with a fluorescence emission peak at 530 nm and green fluorescence upon excitation. 2. Electrostatic attraction, where the positively charged AO binds to the negatively charged phosphate groups of single-stranded nucleic acids through electrostatic attraction. This binding mode primarily occurs with RNA, with a fluorescence emission peak at 640 nm and red fluorescence upon excitation. Small amounts of binding can result in orange or red fluorescence. Therefore, acridine orange appears green when embedded in double-stranded DNA, and emits orange or red fluorescence when bound to single-stranded DNA or RNA. AO staining solution should be diluted to an appropriate concentration before use. After staining, observation under a fluorescence microscope reveals that AO can penetrate normal cell membranes, causing the cell nucleus to exhibit a uniform green or yellow-green fluorescence. In apoptotic cells, chromatin condenses or breaks into fragments of varying sizes, forming apoptotic bodies. AO stains these cells with dense, intense yellow-green fluorescence or yellow-green fragments, while the yellow fluorescence of necrotic cells weakens or even disappears. AO staining is often combined with EB staining for double staining, as EB only stains dead cells, causing them to produce orange-yellow fluorescence. This allows for the distinction between normal, apoptotic, and necrotic cells. |
| General Notes | 1. This product does not contain a membrane permeabilizing agent and is rarely used alone. 2. AO staining is often used in combination with EB staining to distinguish normal cells from apoptotic cells and necrotic cells. 3. Centrifugation in a low-temperature centrifuge is more effective. 4. During operation, be careful to minimize exposure of the reagent to strong light. 5. For your safety and health, please wear a lab coat and disposable gloves during operation. |
| Concentration | 1mg/ml |
| Storage Temp. | 2-8℃, protect from light, valid for 12 months. |
