Validity: One year.

Product introduction:

Apoptosis is one of the basic characteristics of cells. It plays a very important role in the body's embryonic development, tissue repair, and internal environment stability.In normal cells, phosphatidylserine (PS) is only distributed inside the lipid bilayer of the cell membrane, while in the early stage of apoptosis, the phosphatidylserine in the cell membrane Acid (PS) turns from the inside to the outside of the lipid membrane. Annexin Ⅴ is a Ca 2+ -dependent phospholipid binding protein with a molecular weight of 35-36kD , which can interact with apoptosis PS with high affinity and specific binding in the middle flipped out of the membrane.

Use Annexin Ⅴ labeled with EGFP as a fluorescent probe, and use flow cytometry or fluorescence microscopy to detect the occurrence of apoptosis, normal cells and The cell membrane of early apoptotic cells is intact. Propidium iodide (PI) is a nucleic acid dye, it can not penetrate the intact cell membrane, but is in apoptosis In the middle and late cells and dead cells, PI can penetrate the cell membrane and combine with the nucleus to appear red. Matching Annexin Ⅴ with PI can distinguish the early apoptosis cells from the late cells and dead cells. On the scatter plot of a dual-variable flow cytometer, the lower left quadrant shows live cells, as(EGFP-/PI-); the upper right quadrant is non-viable cells, ie necrotic cells, (EGFP+ /PI+ ); and the lower right quadrant is apoptotic cells, showing (EGFP+ /PI-).

Instructions:

➢ Sample dyeing:

1. Centrifuge to collect suspended cells, centrifuge speed 2000RPM, centrifuge time 5min, discard the medium. (The time for digesting adherent cells with trypsin is not suitable Too long to prevent false positives).

2. Wash the cells twice with cold PBS. (2000RPM, centrifugation time 5min to collect cells).

3. Suspend the cells with 400ul 1X Annexin V binding solution at a concentration of approximately 1 x 106cells/ml.

4. Add 5ul Annexin V-EGFP staining solution to the cell suspension, mix gently, and incubate at 2-8°C in the dark for 15 minutes.

5. After adding 10ul PI staining solution, mix gently and incubate at 2-8°C in the dark for 5 minutes.

6. Use flow cytometer or fluorescence microscope to detect within 1 hour.

➢ Flow cytometry analysis:

The treated cells can now be analyzed on a flow cytometer. The excitation wavelength is 488nm.

Follow the conventional flow-type apoptosis detection procedure. The green fluorescence of Annexin V-EGFP is detected through the FL1 channel; the red fluorescence of PI is passed through FL2 Or FL3 channel detection, FL3 is recommended.

➢ Fluorescence microscope observation:

1. Drop the cell suspension double-stained with Annexin V-EGFP/PI on a glass slide, and cover the cells with a cover glass.

Note: For adherent cells, after staining like suspended cells, drop a drop of cell suspension on a glass slide, cover the cells with a cover glass, and observe under a fluorescent microscope. Can also be straight Coverslips were used to cultivate cells and induce apoptosis. Depending on the size of the coverslip, place them in a 24-well or 12-well cell culture plate and then induce apoptosis.

Cell staining in cells Conducted in the culture plate. First rinse twice with PBS, add 400μl Annexin V binding solution to the well. Then add 5μl Annexin V-EGFP staining solution and 10μl Propidium Iodide staining solution, mix well. Incubate at room temperature for 10 minutes in the dark.

2. Observe with a two-color filter under a fluorescent microscope. Using the FITC filter (blue light) on the fluorescence microscope, the fluorescence signal of Annexin V-EGFP was Green; with Rhodamine filter (green light), the PI fluorescence signal is red, and the cells positive for Propidium Iodide staining will appear in the entire cytoplasm With different intensity of yellow-red, early apoptotic cells will not be stained by Propidium Iodide or show background fluorescence, while necrotic or late apoptotic cells will show It shows a yellow-red cytoplasm, a red nucleus and a green membrane surrounding the cell. Membrane shrinkage and blistering can also be observed in late apoptotic cells.