Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue 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™ Violet 450 working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 405/450 nm (Cutoff = 420 nm) or fluorescence microscope with Violet filter

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

Add 10 μL of 100X Apopxin™ Violet 450 (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Apopxin™ Violet 450 working solution. Note: 100 μL of Apopxin™ Violet 450 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 staurosporine) to induce apoptosis. Note: Some compounds such as camptothecin might give false positive response due to the broad emission spectrum from 380 to 490 nm when excited at 405 nm.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Apopxin™ Violet 450 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 = 405/450 nm (Cutoff = 420 nm) or image cells using fluorescence microscope with Violet filter.