Annexin V FITC / PI Apoptosis Detection Kit
Annexin V-FITC Apoptosis Detection Kit
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 (PS) in the cell membrane turns from the inside to the outside of the lipid membrane. Annexin â…¤ is a Ca2 + -dependent phospholipid binding protein with a molecular weight of 35-36kD, which can specifically bind to PS with high affinity turned over outside the membrane during apoptosis.
Using Annexin â…¤ labeled with FITC as a fluorescent probe, the occurrence of apoptosis can be detected by flow cytometry or fluorescence microscopy. The cell membranes of normal cells and early apoptotic cells are intact. Propidium iodide (PI) is a nucleic acid dye, which can not 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. Matching Annexin â…¤ with PI can distinguish the early apoptosis cells from the late cells and dead cells. On the scatter plot of the dual-variable flow cytometer, the lower left quadrant shows live cells as (FITC- / PI-); the upper right quadrant is non-viable cells, ie necrotic cells, (FITC + / PI +); and the lower right quadrant is Apoptotic cells appear (FITC + / PI-).
product composition:
Product ID
401001
401002
401003
specification
20 assays
50 assays
100 assays
Annexin V-FITC
100μL
250μL
500μL
Propidium Iodide
200μL
500μL
1 mL
1X Binding Buffer
8 mL
20 mL
40mL
Instructions:
1. Collect the suspended cells by centrifugation, rotate the microcentrifuge at 2000 RPM, centrifuge for 5 minutes, and discard the medium.
2. Wash the cells twice with cold PBS.
3. Suspend the cells with 400ul 1X Binding Buffer, the concentration is about 1 X 106 cells / ml.
4. Add 5ul Annexin V-FITC to the cell suspension, mix gently, and incubate at 2-8 ° C in the dark for 15 minutes.
5. After adding 10ul PI, mix gently and incubate at 2-8 ° C in the dark for 5 minutes.
6. Check with flow cytometry or fluorescence microscope 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-FITC is detected through the FL1 channel; the PI red fluorescence is detected through the FL2 or FL3 channel, and FL3 is recommended.
You can refer to the following streaming settings:
Before using flow cytometry to analyze Annexin V-FITC / PI double-stained cells correctly, the instrument's fluorescence compensation is required to remove the superposition between the excitation lights of the two dyes. 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 single-stained by Annexin V and PI at the beginning of the experiment to adjust the fluorescence compensation to remove the spectral overlap. The position of the cross gate was set according to the analysis of the untreated cell blank control and the single-stained control after cell staining with Annexin V and PI, respectively.
1. Load unstained cells, display the cells on a linear FS-SS dot plot and gate the target cell population.
2. Create a two-parameter dot plot of LogFL1-LogFL2 (preferably FL3) and analyze the cells gated in the light scattering diagram; ensure that> 98% of the cells are in the center of the lower left quadrant on the X and Y axis Log 1 as the boundary.
3. Examine annexin V-FITC single-stained cells and check the FL1-FL2 (or FL3) scatter plot to ensure that there are no particles in the upper left and upper right quadrants. If there are particles in the upper quadrant, it indicates that there is leakage of fluorescence; at this time, the fluorescence of FL1 is detected by FL2 (or FL3) PMT. In order to correct this phenomenon, increase the compensation percentage of FL1 leakage to FL2 (or FL3) fluorescence (this may be between 1-5%). If this adjustment does not effectively remove the positive signal of FL2, then the voltage of FL2 (or FL3) PMT should be reduced.
4. Detect PI-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 there are particles in the right quadrant, it indicates that there is fluorescence leakage; at this time, the fluorescence of PI is detected by FL1 PMT. To correct this phenomenon, increase the compensation percentage of FL2 (or FL3) leakage to FL1 fluorescence (this may be between 15-25%). If this adjustment does not effectively remove the positive signal of FL1, then the voltage of FL1 PMT should be reduced.
Note: If the PMT voltage is changed during the above adjustment and compensation process, it is recommended to repeat steps 3 and 4 to ensure that it does not cause excessive fluorescence compensation. Overcompensation can be observed from the phenomenon that positive cells are very close 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 that fall in the middle of the lower quadrant of Log 1 as the border quadrant.
5. To set the position of the cross gate, the method of delineating FL1 and FL2 (or FL3) is as follows:
5.1 Set FL1 scale position: The large group of cells in the lower left quadrant are Annexin V staining negative cells (generally these cells will rise to 2 logarithmic coordinate values ​​in the FL1 axis direction). Set the vertical FL1 ruler to the position 0.1-0.2 Log units to the right of the Annexin V negative population.
5.2 Set the FL2 (or FL3) scale position: You can distinguish the PI + and PI- cell populations by double positive cells with certain data. Under this condition, two groups of cells may be identified, one is located at the lower right of the scatter plot (ANN + / PI-) and the other is at the upper right (ANN + / PI +). The horizontal line can be placed between these two groups of cells. If there is no PI + cell in the analyzed cell population, it is best to distinguish the PI + cell by referring to the double-negative cell population, and set the horizontal line at 0.1-0.3 Log units above the double-negative cell.
Note: Cells outside the gate of the negative population can be recognized as Annexin V or Annexin V and PI positive cells.
Fluorescence microscope observation:
1. Drop a drop of cell suspension double-stained with Annexin V-FITC / PI on a glass slide and cover the cells with a cover glass.
Note: For adherent cells, coverslips can be used to directly culture the cells and induce apoptosis.
2. Observe with a two-color filter under a fluorescent microscope. Annexin V-FITC fluorescence signal is green, PI fluorescence signal is red.
Storage conditions:
Store at 2-8 ° C.
Validity period:
One year.
Precautions:
1. Before opening the cap, please centrifuge the reagents in the capped centrifuge tube briefly, and flick the liquid on the inner wall of the cap to the bottom of the tube to avoid liquid spillage when opening the cap.
2. Cell handling requires careful operation, try to avoid artificial damage to cells.
3. Annexin V-FITC and Propidium iodide are light-sensitive substances, please avoid light during operation.
4. Successful detection of apoptosis is affected by several factors, such as cell type, density of PS on the cell membrane, the proportion of PS flipping when apoptosis occurs, the method of inducing apoptosis, the reagents used, the time of inducing apoptosis, etc. Optimizing these influencing factors is crucial to the success of the experiment.
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