Cell Meter™ Annexin V Apoptosis Assay Kit

Protocol summary

1. Prepare cells with test compounds (200 µL/sample) 
2. Add APC-Annexin V assay solution
3. Incubate at room temperature for 20 - 60 minutes
4. Analyze cells using flow cytometer with 660/20 nm filter (APC channel)

Important notes
Thaw 100X Propidium Iodide (Component C) at room temperature before starting the experiment.

1. APC-Annexin V stock solution (100X):
Add 200 µL PBS with 0.2% BSA into the vial of APC-Annexin V conjugate (Component A) and mix well to make 100X APC-Annexin V stock solution. Note: Store the reconstituted 100X APC-Annexin V stock solution at 4 ^oC. Do Not Freeze.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with test compounds for a desired period of time (4-6 hours for Jurkat cells treated with staurosporine) to induce apoptosis. Note: Annexin V flow cytometric analysis on adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.
    
2. Centrifuge the cells to get 1-5 × 10⁵ cells/tube.
   
   
3. Resuspend cells in 200 µL of Assay Buffer (Component B).
   
   
4. Add 2 µL of 100X APC-Annexin V stock solution into the cells.
   
   
5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells for necrosis cells.
   
   
6. Incubate at room temperature for 20 to 60 minutes, protected from light.
   
   
7. Optional: Add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.
   
   
8. Monitor the fluorescence intensity of APC-Annexin V using a flow cytometer with 660/20 nm filter (APC channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells.