Annexin V is a reagent for detecting cell apoptosis. In normal cells, phosphatidylserine is only distributed on the inner side of the lipid bilayer of the cell membrane. In the early stage of cell apoptosis, the membrane phosphatidylserine (PS) is turned from the inner side of the lipid membrane to outside. As a phospholipid-binding protein, Annexin V has a high affinity to phosphatidylserine, and it binds to the cell membrane of early apoptosis cells through the phosphatidylserine exposed on the outside of the cell. Therefore, Annexin V is a sensitive indicator for detecting early cell apoptosis.
Apoptosis is one of the basic characteristics of cells, and it plays a very important role in the embryonic development, tissue repair, and stability of the internal environment of the body. In normal cells, phosphatidylserine (PS) is only distributed on the inner side of the lipid bilayer of the cell membrane, but in the early stage of apoptosis, the phosphatidylserine (PS) in the cell membrane is turned from the inside to the outside of the lipid membrane. Annexin Ⅴ is a Ca-dependent phospholipid-binding protein with a molecular weight of 35-36kD, which can specifically bind with high affinity to PS that is turned over to the outside of the membrane during apoptosis.
Using FITC-labeled AnnexinⅤ as a fluorescent probe, the occurrence of apoptosis can be detected by flow cytometry or fluorescence microscopy, and the cell membranes of normal cells and early apoptotic cells are intact. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the complete cell membrane, but in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and combine with the nucleus to appear red. The matching of Annexin Ⅴ and PI can distinguish early-stage apoptosis cells from late-stage cells and dead cells.
Collect the suspended cells by centrifugation, the speed of the microcentrifuge is 2000RPM, the centrifugation time is 5min, and the medium is discarded;
Wash the cells twice with cold PBS;
Suspend the cells with 400ul 1X Binding Buffer, the concentration is about 1 X 10 cells/ml;
Add 5ul Annexin V-FITC to the cell suspension, mix gently and incubate at 2-8°C for 15 minutes in the dark. 5. After adding 10ul PI, mix gently and incubate at 2-8°C in the dark for 5 minutes;
Detection by flow cytometry or fluorescence microscopy within 1 hour.
Treated cells are now ready for analysis on a flow cytometer.
Fluorescence compensation of the instrument is required to remove the overlap between the excitation light of the two dyes before using the flow cytometer to correctly analyze Annexin V-FITC/PI double-stained cells. Because the fluorescence compensation setting is directly related to the voltage of the PMT, the compensation varies between different instruments. It is recommended to analyze the cells stained with Annexin V and PI at the beginning of the experiment to adjust the fluorescence compensation to remove spectral overlap. The position of the cross gate was set according to the analysis of the blank control of untreated cells and the single-stained control after the cells were stained with Annexin V and PI respectively.
1. Load unstained cells, visualize the cells on a linear FS-SS dot plot and gate the target cell population.
2. Establish a LogFL1-LogFL2 (preferably FL3) dual-parameter dot plot and analyze the gated cells in the above light-scattering plot; ensure that >98% of the cells are in the central area of the lower left quadrant bordered by Log 1 on the X and Y axes.
3. Detect annexin V-FITC single-stained cells and check the scatter plot of FL1-FL2 (or FL3) to ensure that there are no particles in the upper left and upper right quadrants. Fluorescence leakage is indicated if particles appear in the upper quadrant; FL1 fluorescence is detected by the FL2 (or FL3) PMT. To correct this phenomenon, increase the compensation % of FL1 leakage to FL2 (or FL3) fluorescence (this may be between 1-5%). If this adjustment does not effectively remove the FL2 positive signal, then reduce the FL2 (or FL3) PMT voltage.
4. Detect PI single-stained cells and check the FL1-FL2 (or FL3) scatter plot to ensure that there are no particles in the upper right and lower right quadrants. If particles appear in the right quadrant, there is fluorescence leakage; at this time, the fluorescence of PI is detected by FL1 PMT. To correct this phenomenon, increase the compensation % of FL2 (or FL3) leakage to FL1 fluorescence (this may be between 15-25%). If this adjustment does not effectively remove the FL1-positive signal, then reduce the voltage on the FL1 PMT.
Note: If the voltage of the PMT is changed during the above adjustment and compensation process, it is recommended to repeat steps 3 and 4 to ensure that excessive fluorescence compensation is not caused. Overcompensation can be observed from the close proximity of positive cells to the target. A proper compensation should be that the fluorescence intensity of single positive cells is consistent with the fluorescence intensity of negative cells in the middle of the lower left quadrant where Log 1 is the boundary.
5. Set the position of the cross gate, the delineation method of FL1 and FL2 (or FL3) is as follows:
5.1 Setting the position of the FL1 scale: The large group of cells in the lower left quadrant are cells negative for Annexin V staining (generally these cells will rise to 2 logarithmic coordinates in the direction of the FL1 axis). Set the vertical FL1 scale at 0.1-0.2 Log units immediately to the right of the Annexin V negative population.
5.2 Set the position of FL2 (or FL3) scale: PI+ and PI- cell populations can be distinguished by a certain number of double-positive cells. Two populations of cells may be identified under this condition, one located in the lower right of the scatter plot (ANN+/PI-) and the upper right (ANN+/PI+). A horizontal line can be placed in the middle of these two groups of cells. If there are no PI+ cells in the analyzed cell population, it is best to distinguish PI+ cells by reference to the double negative cell population, setting the horizontal line at 0.1-0.3 log units above the double negative cells.
Note: Cells outside the negative population gate can be identified as Annexin V or Annexin V and PI positive cells.
Fluorescence microscope observation:
Put a drop of cell suspension double-stained with Annexin V-FITC/PI on a glass slide, and cover the cells with a coverslip.
NOTE: For adherent cells, cells can be cultured directly with coverslips and apoptosis induced.
Observe under a fluorescence microscope with a dichroic filter. The fluorescence signal of Annexin V-FITC is green, and the fluorescence signal of PI is red.
1. Before opening the cap, please centrifuge the reagent in the screw-cap centrifuge tube briefly, and shake the liquid on the inner wall of the cap to the bottom of the tube to avoid liquid spilling when opening the cap.
2. Cell handling requires careful operation to avoid artificial damage to cells.
3. Annexin V-FITC and Propidium iodide are photosensitive substances, please avoid light when handling.
4. Successful detection of apoptosis is affected by the following factors, such as cell type, density of PS on cell membrane, proportion of PS turnover when apoptosis occurs, method of inducing apoptosis, reagent used, time of inducing apoptosis, etc. , optimizing these influencing factors is crucial to the success of the experiment.