Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds (200 µL/sample)
2. Add Annexin V-FITC assay solution
3. Incubate at room temperature for 30 - 60 minutes
4. Analyze cells with a flow cytometer using FL1 channel (Ex/Em = 490/525 nm)

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

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 2 - 5 × 10⁵ cells/tube.
   
   
3. Resuspend cells in 200 µL of Assay Buffer (Component B).
   
   
4. Add 2 µL of Annexin V-FITC (Component A) into the cells.
   
   
5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) for necrosis cells.
   
   
6. Incubate at room temperature for 30 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 Annexin V-FITC using a flow cytometer with FL1 channel (Ex/Em = 490/525 nm). Measure the cell viability using FL2 channel when propidium iodide is added into the cells.