Product Introduction:

Early apoptotic changes occur on the surface of the cell membrane, and one of these changes on the surface of the cell membrane is the transfer of phosphatidylserine (PS) from inside the cell membrane to outside the cell membrane, exposing PS to the outer surface of the cell membrane. PS is a negatively charged phospholipid, which normally exists in the inner side of the cell membrane. During cell apoptosis, the asymmetry of this phospholipid distribution on the cell membrane is destroyed, and PS is exposed to the outside of the cell membrane. Annexin V has easy binding to phospholipids such as PS and has a high affinity for PS. Thus, the protein can act as a sensitive probe to detect PS exposed on the surface of the cell membrane. The transfer of PS to the outside of the cell membrane is not unique to apoptosis and can also occur in cell necrosis. The difference between the two modes of cell death is that the membrane is intact in the initial stage of apoptosis, while the integrity of the membrane is destroyed in the early stages of cell necrosis. Therefore, the Annexin V and PI double staining method can be used to detect the early apoptosis of cells by flow cytometry.

Operation steps :

1, cell sample preparation:

a) For adherent cells:

Carefully collect the cell culture solution into a centrifuge tube for later use. Digest the cells with pancreatic enzymes without EDTA, and when the cells can be gently blown down by pipette or gun, add the cell culture solution collected earlier, blow down all the adherent cells, and gently blow away the cells. Collected again into the centrifugal tube. Centrifuge at 1000rpm for 5min and precipitate the cells. For specific cells, if the cells cannot be completely centrifuged to the bottom of the centrifuge tube, the centrifuge time can be extended appropriately or the centrifugal force can be slightly increased. Carefully remove the supernatant, leaving about 50μl of culture solution to avoid cell absorption. Add about 1ml PBS precooled at 4℃, suspend the cells, centrifuge the precipitated cells again, and carefully remove the supernatant.

b) For suspended cells: Centrifuge at 1000rpm for 5min and precipitate cells. For specific cells, if the cells cannot be completely centrifuged to the bottom of the centrifuge tube, the centrifuge time can be extended appropriately or the centrifugal force can be slightly increased. Carefully remove the supernatant, leaving about 50μl of culture solution to avoid cell absorption. Add about 1ml PBS precooled at 4℃, suspend the cells, centrifuge the precipitated cells again, and carefully remove the supernatant.

2. Dilute the binding buffer with deionized water by 1:3 (4ml 4× binding buffer +12ml deionized water);

3. The cells were re-suspended with 1x binding buffer and the concentration was adjusted to 1-5×10⁶/ml;

4. 100μl cell suspension was placed in a 5ml flow tube, mixed with 5μL Annexin V/Alexa Fluor 488, and incubated at room temperature for 5 minutes away from light.

5. Add 10μl of 20ug/ml Propium iodide solution (PI) and add 400μl PBS for immediate flow detection.

Experimental design:

1. Blank tube: cells of negative control group without Annexin V/Alexa Fluor 488, propyl iodide solution (PI). Used to regulate voltage;

2, single stain tube: positive control cells, only Annexin V/Alexa Fluor 488. For adjusting compensation;

3. Test tube: Treated cells, Annexin V/Alexa Fluor 488, Propyl iodide solution (PI). Adjust the voltage compensation with blank tube and single dye tube

, the required streaming data is obtained.

Note:

1, Annexin V is binding to phosphatidylserine (PS), and PS does not differ between species. In normal cells, PS only distributed in the inside of the lipid bilayer of the cell membrane, but in the early stage of apoptosis, PS turned from the inside of the lipid membrane to the outside.

2. Digestion of pancreatic enzyme at low concentration, gentle blowing of adherent cells 2 to 3 times, centrifuge at 4℃1000rpm for 5min centrifuge, if properly treated, damage caused by pancreatic enzyme can be controlled within 5%, and the experimental results will not be significantly affected when there is a control group.

3. It is not only difficult to judge whether the staining of each group is uniform and sufficient, but also PI itself is toxic to cells and has a greater impact on the experimental results than pancreatic enzyme, so it is not recommended to do so;

4. Annexin V is a CA-dependent protein, so EDTA cannot be added to prevent EDTA from chelating Ca ions and thus affecting Annexin V, thus affecting the results.

5. When apoptosis is detected by flow cytometry, PI is greatly affected by time, because the labeling of PI will increase cytotoxicity, and the staining of PI will increase with time, especially in the detection of early apoptosis, if the time is prolonged, the error will be significantly increased in addition to the widening of the cell group gap on flow cytometry. Generally, PI is added immediately after the machine, and then the detection is completed within an hour. Both methods can be used, but the error caused by following our steps will be smaller.

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