Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red 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™ Deep Red working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 650/680 nm (Cutoff = 665 nm) or fluorescence microscope with Cy5 filter

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

Add 10 μL of Apopxin™ Deep Red (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Apopxin™ Deep Red working solution. Note: 100 μL of Apopxin™ Deep Red 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™ Deep Red 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 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 = 650/680 nm (Cutoff = 665 nm) or image cells using a fluorescence microscope with Cy5^® filter.