Annexin V-Cy5/SYTOX Green Apoptosis Detection Kit

This plan is only a guide, please modify it according to your specific needs.

I. Cell suspension staining:

i. Suspension cell staining

1. Centrifuge the cells at 1000g for 5 minutes, discard the supernatant, gently resuspend the cells in an appropriate amount of PBS and count.

Note: Resuspension of cells in PBS also plays a role in washing the cells to ensure that the subsequent binding of Annexin V-Cy5 is not interfered with. This step cannot be omitted.

2. Take about 5-10×10⁴ cells, centrifuge at 1000g for 5 minutes, discard the supernatant, and add 195μl Annexin V Binding Buffer to gently resuspend the cells.

3. Add 5μl Annexin V-Cy5 and 1μl SYTOX Green staining solution and mix gently.

4. Incubate at room temperature (20-25℃) away from light for 10-20 minutes.

Note: Cells can be resuspended 2-3 times during incubation to improve staining results.

5. Transfer the sample to an ice bath. It is recommended to use aluminum foil to protect it from light.

6. Use flow cytometry or fluorescence microscopy to detect the staining results.

ii. For post-digestion detection of adherent cells:

1. (Important step) Aspirate the cell culture medium and transfer it to a centrifuge tube of appropriate size.

2. (key step) Use PBS to gently wash the adherent cultured cells once, add an appropriate amount of trypsin cell digestion solution to digest the cells, and incubate at room temperature until the cells gradually become round and loose, and the cells appear when they are shaken gently. After falling off, immediately add the cell culture medium collected in step 1 to the digested cells to terminate digestion, and gently pipet down the cells.

Notice:

①  For collecting apoptotic or necrotic cells suspended in the culture medium, it is recommended to use the cell culture medium collected in step 1 to terminate digestion in this step.

②  Trypsin digestion needs to be appropriate. If the digestion time is too short, the cells need to be blown hard to fall off, which is easy to cause cell membrane damage, resulting in false positives of cell necrosis. If the digestion time is too long, it is also easy to cause cell membrane damage and false positives, and even affect the binding of phosphatidylserine on the cell membrane with Annexin V-Cy5, thereby interfering with the detection of cell apoptosis.

③  Try to avoid EDTA in the trypsin digestion solution. Annexin V is a Ca²⁺-dependent protein. EDTA can chelate Ca²⁺, thereby interfering with the binding of Annexin V and phosphatidylserine (PS), affecting the experimental results.

④  If you use trypsin containing EDTA to digest cells, it is recommended to wash the cells several times with PBS after digestion and before adding Annexin V-Cy5 to remove the effect of EDTA on apoptosis detection.

3. Transfer the cell solution to a centrifuge tube, centrifuge at 1000g for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count.

Note: Try to remove trypsin as much as possible in this step. The remaining trypsin will digest and degrade Annexin V-Cy5 added subsequently, affecting the staining effect.

4. Take about 5-10×10⁴ cells, centrifuge at 1000g for 5 minutes, discard the supernatant, add 195μl Annexin V Binding Buffer and gently resuspend the cells.

5. Add 5μl Annexin V-Cy5 and 1μl SYTOX Green staining solution, mix gently, and incubate at room temperature (20-25℃) in the dark for 10-20 minutes.

Note: Cells can be resuspended 2-3 times during incubation to improve staining results.

6. Transfer the sample to an ice bath. It is recommended to use aluminum foil to protect it from light.

7. Use flow cytometry or fluorescence microscopy to detect the staining results.

II. In situ fluorescence detection of adherent cells:

1. (Optional) If conditions permit, it is recommended to centrifuge the multi-well plate at 1000g for 5 minutes after the induction of apoptosis.

2. Aspirate away the cell culture medium, add PBS and wash once. If conditions permit, it is recommended to centrifuge the multi-well plate at 1000g for 5 minutes before aspirating and discarding the PBS.

3. Add 195μl Annexin V Binding Buffer.

4. Add 5μl Annexin V-Cy5 and 1μl SYTOX Green staining solution, mix gently, and incubate at room temperature (20-25°C) away from light for 10-20 minutes.

5. Transfer the sample to an ice bath. It is recommended to use aluminum foil to protect it from light.

6. Use a fluorescence microscope to detect the staining results.

Precautions:

1. Cells should be tested as soon as possible after staining. It is recommended to complete the test within 1 hour. It is not recommended to store cells in the binding solution for too long.

2. If it is used for flow cytometry detection, it can be tested immediately. Annexin V-Cy5 is a red fluorescent protein with a maximum excitation/emission wavelength of 650/670nm. SYTOX Green is a green fluorescent protein with a maximum excitation/emission wavelength of 504/523nm.

3. After staining, you can directly test the cells without changing the staining solution. If you cannot test immediately, it is recommended to centrifuge to remove the staining solution and resuspend the cells in PBS or Annexin V Binding Buffer.

4. If the cells are detected under a fluorescence microscope after suspension staining, it is recommended to collect the cells by centrifugation at 1000g for 5 minutes, gently resuspend the cells in 50-100μl Annexin V Binding Buffer, and observe under a fluorescence microscope after smearing.

5. When using the flow cytometer for detection for the first time, it is recommended to set up three control groups: unstained, SYTOX Green single-stained and Annexin V-Cy5 single-stained.

6. When using a flow cytometer for detection, if you find that too many SYTOX Green false-positive cells appear when Annexin V-Cy5 is stained alone, and it cannot be improved by adjusting relevant settings and parameters, you can use PBS to add Annexin V-Cy5. Dilute 3-10 times before testing.

7. This product is only suitable for double staining of apoptosis in cell models. It is also suitable for detecting sperm apoptosis, but cannot be used to detect apoptosis in tissue samples.

8. Annexin V-Cy5 labels apoptotic cells by combining phosphatidylserine (PS). PS is relatively conserved among different species. Therefore, this product is suitable for apoptosis detection of cells of different species. For relevant experimental steps, please refer to relevant literature.

9. This product can be used to stain cells together with antibodies, but it is recommended to stain them separately. It is recommended to replace the buffer with Annexin V Binding Buffer for apoptosis detection after antibody staining.

10. If the sample comes from blood, platelets in the blood must be removed. PS in platelets will interfere with the experimental results.

11. The nerve cell membrane is easily damaged and everted, resulting in false positives. Therefore, this product is not suitable for detecting nerve cell apoptosis.

12. Be sure to be gentle during operation, and confirm whether there are cells at the bottom of the tube after centrifugation.

13. Fluorescent dyes all have quenching problems. Please try to avoid light during the experiment to slow down fluorescence quenching.

14. For your safety and health, please wear a lab coat and disposable gloves.