Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds (200 µL/sample) 
2. Add Apopxin™ Green assay solution
3. Incubate at room temperature for 20 - 60 minutes
4. Analyze cells using flow cytometer with 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 camptothecin) to induce apoptosis. Note: Apopxin™ binding 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 Apopxin™ Green (Component A) 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 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.