| Usage | 1. Instrument parameter adjustment
1. Collect 1 × 106-3×106The cells were washed twice by centrifugation with pre-cooled PBS and the supernatant was discarded.
2. Add 500uL of Apoptosis Positive Control Solution to resuspend and incubate on ice for 30 minutes.
3. Wash by centrifugation with pre-cooled PBS and discard the supernatant.
4. Add an appropriate amount of pre-cooled 1 × Binding Buffer to resuspend, and add the same number of untreated viable cells to mix with it. Add pre-cooled 1 × Binding Buffer to replenish to 1.5 mL, and divide it into three tubes equally, of which one tube is a blank control tube and two tubes are single dye tubes.
5. Add 5 uL of Annexin V-PE or 10 uL of 7-AAD to the single stain tube and incubate for 5 minutes at room temperature and protect from light.
6. On the flow cytometer, the voltages of FSC, SSC and fluorescence channel were adjusted with a blank tube, and under this voltage condition, the fluorescence was adjusted with a single staining tube
Compensation of channels.
Note:Certain processes of treating adherent cells as single cells cause cell membrane damage, resulting in false positives for Annexin V. Therefore, optimization is needed. It is recommended to treat adherent cells with enzymes that are more gentle on cells, such as Accutase.
The adherent cells can be cultured on a suitable vessel, such as a sterile glass slide, and stained with suitably modified methods. However, this method has not been tested in our laboratory.
2. Sample testing
1. Induce apoptosis according to the experimental protocol.
2. Centrifuge washing with pre-cooled PBS and collect 1-10 × 105Cells include cells in the culture supernatant. Dilute 5 × Binding Buffer into 1 × working solution with double distilled water, and take 500 uL of 1 × Binding Buffer to resuspend the cells.
3. Add 5 uL Annexin V-PE and 10 uL 7-AAD per tube.
4. After gently vortex and mix well, incubate at room temperature and protect from light for 5 minutes.
5. Get on the computer for analysis. |
| Beilstein | 0 |
| Synonym | Annexin V-PE/7-AAD Apoptosis Analysis Kit |
| Description | Annexin V (or Annexin A5) is a member of the annexin family of intracellular proteins that binds to phosphatidylserine (PS) in a calcium-dependent manner. PS is present in the inner lining of the plasma membrane of normal cells, but in the early stage of apoptosis, membrane asymmetry is lost and PS translocates to the cell surface. Fluorescently Labeled Annexin V
Can specifically bind to it, indicating that the cell is an apoptotic cell. This kit takes only 15 minutes to stain, which is quick and easy. Kit Components: | Components | 25T | 100T | | Annexin V-PE | 150uL | 500uL | | 7-AAD | 300uL | 1mL | | 5 × Binding buffer | 5mL | 15mL | | Apoptosis Positive Control Solution | 5mL | 5mL |
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| PubChem CID | 0 |
| General Notes | 1. Please read the instructions carefully before using this product. This product is for scientific research only, not diagnostic.
2. For your safety and health, please wear experimental protective clothing, gloves, masks and other necessary protective equipment. |
| Storage Temp. | Store at 2-8 °C, do not freeze, shelf life 1 year. |
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