Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Green Fluorescence Optimized for Microplate Readers*

Protocol summary

1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
2. Add equal volume of Apopxin™ Green working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) or fluorescence microscope with FITC filter

Important notes
Warm Assay Buffer (Component B) at room temperature before starting the experiment.

Add 10 µL of  100X Apopxin™ Green (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Apopxin™ Green working solution. Note: 100 µL of Apopxin™ Green working solution is enough for one well. Prepare fresh before use.

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

1. Treat cells with desired test compounds by adding 10 µL/well (96-well plate) of 10X test compound solution into cell preparation buffer, for a total volume of 100 µL/well. For a 384-well plate, use 5 µL/well of 5X test compound solution into cell preparation buffer, for a total volume of 25 µL/well. For blank wells (medium without the cells), add the same amount of compound buffer.
   
   
2. Incubate the cell plate in a 5% CO₂, 37°C incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Apopxin™ Green working solution into each well.
   
   
4. Incubate the cell plate at room temperature for at least 1 hour, protected from light.
   
   
5. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
   
   
6. Monitor the fluorescence intensity with a fluorescence microplate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) or image cells using fluorescence microscope with FITC filter.