1. The cells to be stained for analysis were washed twice with cold PBS and resuspended at a concentration of 1 x 106 cells/mL in an appropriate binding buffer. (staining buffer, binding buffer, must contain calcium ions)
2. Pipette 100 ul of cells (1 x 105) into the tube. (must be done at room temperature)
3. Add the appropriate amount of fluorescently labeled annexin V reagent and PI. (must be done at room temperature)
4. Incubate and incubate for 15 minutes at room temperature. (must be done at room temperature)
5. Add 400 ul of staining buffer after incubation and immediately analyze by flow cytometry. (The experiment must be completed in an hour, otherwise it is meaningless!)
We recommend the preparation of three additional quality control samples to set the fluorescence compensation of the flow cytometer and set the range of the cross gate:
a) cells that are not stained;
b) cells stained with only fluorescently labeled annexin V;
c) cells stained only with PI.
Annexin V and PI double positives indicate that the cell population contains dead cells. We recommend that one cell line can be used to prepare the above three control tubes for use as annexin V and PI positive staining controls. Human U-937 cells incubated for 2-3 hours with RPMI + 10% FCS + 2-4 ng/mL of TNF-a, or 1-2 hours of mouse lymphocytes in culture may be used as control cells.
Please set the instrument compensation correctly according to the following suggestions. The following protocols can be modified slightly depending on the cell type.
Analysis of stained cells on a flow cytometer:
Correct analysis of annexin V-FITC and PI double-labeled cells using flow cytometry requires fluorescence compensation of the instrument to remove the superposition of the excitation light of the two dyes. Because the fluorescence compensation settings are directly related to the voltage of the PMT, the compensation between the different instruments is different. We propose to analyze single-stained cells at the beginning of the experiment to adjust the fluorescence compensation to remove spectral overlap.
1. Load the unstained cells, display the cells on the linear FS-SS dot plot and set the gate to the target cell population.
Note: The light scattering signal changes after the cells are cultured. Care should be taken to use a side-scattering light gate to identify these altered cells.
2. Establish the LogFL1-LogFL2 two-parameter dot plot and analyze the cells in the above light scattering map; ensure that >98% of the cells are in the lower left quadrant central region with the X and Y axes Log 1 as the boundary.
3. Detect annexin V-FITC single stained cells and examine the FL1-FL2 scatter plot to ensure no particles in the upper left and upper right quadrants. If particles appear in the upper quadrant, there is fluorescence leakage; at this time, the fluorescence of FL1 is detected by FL2 PMT. To correct this, increase the compensation for FL1 leakage to FL2 fluorescence (this may be between 1-5%). If this adjustment does not effectively remove the positive signal of FL2, then the voltage of FL2 PMT is reduced.
4. Detect PI-stained cells and examine the FL1-FL2 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, increase the % of FL2 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 is reduced.
5. If the PMT voltage is changed during the above adjustment compensation process, we recommend repeating steps 3 and 4 to ensure that no excessive fluorescence compensation is caused. Overcompensation can be observed from the fact that positive cells are very close to the standard. A proper compensation should be that the fluorescence intensity of a single positive cell is consistent with the fluorescence intensity of a negative cell that falls in the lower left of Log 1 as the boundary quadrant.
6. Because a number of annexin V and PI double positive cells are present in many untreated cell populations, the use of the annexin V kit to detect apoptosis must subtract these previously present double positive cells from the treated cells.
7. Set the position of the cross door according to the results of analysis on the flow cell by untreated or control cells stained with annexin V and PI. The method of delineating FL1 and FL2 is as follows:
a. Setting the FL1 scale position: Large groups of cells in the lower left quadrant are cells that are negative for annexin V staining (generally these cells will rise to 2 logarithmic values ​​in the FL1 axis). Set the vertical FL1 scale to the nearest 0.1-0.2 Log unit to the right of the annexin V negative population.
b. Setting the FL2 scale position: The PI+ and PI- cell populations can be distinguished by double positive cells with certain data. Under these conditions, two groups of cells may be identified, one at the bottom right of the scatter plot (ANN+/PI-) and the upper right (ANN+/PI+). The horizontal line can be placed in the middle of the two groups of cells. If there are no PI+ cells in the analyzed cell population, it is best to distinguish PI+ cells from the double negative cell population and place the horizontal line at 0.1-0.3 Log units above the double negative cells. Ideally, the gated method described above can be applied to any cell population to set the position of the cross door.
8. The treated cells can now be stained with annexin V and PI and analyzed on a flow cytometer. Those cells outside the negative population can be identified as annexin V or annexin V and PI positive cells.
Note: It is recommended that cells be stained for analysis as soon as possible. Although cells fixed with 1% formaldehyde are not recommended here, it can be used as a positive control for annexin V and PI double staining in some experiments. The above control was prepared to resuspend the cells in a 1X mixture of staining buffer and fixative. However, this step significantly reduces the staining fluorescence intensity of annexin V and PI.
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