Annexin V-APC/7-AAD Apoptosis Detection Kit

Annexin V, also known as Annexin A5, is a type of protein that belongs to the annexin family and interacts with phosphatidylserine (PS) in a calcium-dependent manner. PS is typically found only on the intracellular surface of the plasma membrane in healthy cells. However, during the beginning stages of apoptosis, membrane asymmetry is lost, and PS moves to the external surface of the plasma membrane. This is where Annexin V comes into play, as it can be labeled with a fluorochrome and used to specifically target and identify apoptotic cells. To obtain accurate results, Annexin V Binding Buffer is the recommended solution to use with Annexin V staining. In addition, to assess viability, 7-AAD Viability Staining solution can be used. Other products available for use in apoptosis studies include Apoptosis Positive Control Solution.

For quality control purposes, every batch of this reagent undergoes testing through immunofluorescent staining followed by flow cytometric analysis. To ensure accurate results, it is recommended to use approximately 5 µl of the reagent for every 10^5 cells. However, it is important to note that the appropriate dilutions may vary depending on the specific application. Therefore, it is necessary to determine the optimal dilution for individual use.

Suggested Staining Protocol:
A.Parameters regulation
1. Harvest cell(1×10⁶-3×10⁶，。，。PBS，。，。
To prepare for future use, take 200μL of 1× binding buffer and suspend a portion of cells in it. Keep the mixture stored at 4° C until needed. This step is crucial for effective experimentation.
To prepare the cells for the apoptosis assay, first, divide them into two groups. For one group, suspend the cells in 500μL Apoptosis Positive Control Solution and let them incubate at room temperature for 10 minutes. The other group should be suspended in 200μL 1× binding buffer. Next, wash the cells in both groups with more than 3.0 mL cold PBS and blot the supernatant. Finally, make sure to thoroughly suspend the cells in the binding buffer before proceeding with the assay.
Combine the two sets of cells and divide them into three tubes, adding 100 μL of cells to each tube. Mix thoroughly before proceeding.
Here is a rearranged version of the given content:
We have three tubes for the experiment. The first tube is the Blank Control, where no additional reagents are added. In the second tube, we add 5μL of Annexin V-APC. Finally, in the third tube, we add 5 μL of 7-AAD solution.
To activate the contents of each tube, gently vortex them prior to incubation. Leave the tubes to incubate for 5 minutes at room temperature, making sure they are protected from any light exposure.
To ensure accurate analysis by flow cytometry, it is important to regulate voltage and compensation using a blank control and single dye sample first. This can be done by following the steps shown in Figure Parameters regulation. By properly adjusting these settings, you will be able to obtain reliable data that can be used to make meaningful conclusions about your samples. Taking the time to perform these preliminary steps can save you a lot of time and frustration later on, so it is well worth the extra effort.
B.Sample detection
To prepare for 10 tests, you need to dilute 6 mL of 5× binding buffer with 24 mL of distilled water. This will ensure that the solution is properly prepared and ready for use in your experiments. It's important to follow these instructions carefully to ensure accurate results and avoid any errors in your testing process. So, be sure to measure your amounts accurately and mix them thoroughly before proceeding with your experiments.
2. Harvest cell(about 1×10⁶cells per test)then wash with cold PBS.
First, centrifuge the cells at 300×g for 10 minutes. Next, suspend the resulting cell pellet in 1 mL of 1× Binding Buffer. Finally, remove the Binding Buffer from the cell pellet to complete the process.
Please adjust the cell concentration to 1x10 and resuspend the cells in 1 mL of 1x Binding Buffer. This process will help ensure the cells are properly prepared for further analysis. Make sure to follow the original text instructions for best results.⁶cells/mL.
5. Add 100 μL of cells (1×10⁵cells) to each labeled tube.
6. Add 5 μL of Annexin V-APC to appropriate tubes.
7. Gently vortex each tube and incubate for 10 minutes in room temperature, protected from light.
8. Add 5 μL 7-AAD solution incubation for 5min in room temperature, protected from light.
9. Add PBS to 500μL and vortex gently.
10. Analyze by flow cytometry in 1 hour.

Keep as concentrated solution. Store at 4°C and protected from prolonged exposure to light. Do not freeze.The validity period is 6 months.