Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Orange 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™ Orange working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or fluorescence microscope with Cy3 filter

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

Add 10 μL of 100X Apopxin™ Orange (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Apopxin™ Orange working solution. Note: 100 μL of Apopxin™ Orange 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 test compounds by adding 10 µL/well (96-well plate) or 2.5 µL/well (384-well plate) of 10X test compound stock solution into PBS or the desired buffer. 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™ Orange 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 a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or image cells using fluorescence microscope with Cy3^® filter.