Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit II *Triple Fluorescence Colors*

Protocol summary

1. Prepare cells with test compounds (200 µL/sample)
2. Add Apopxin™ Deep Red assay solution
3. Incubate at room temperature for 30 - 60 minutes
4. Analyze cells with a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel) or fluorescence microscope with DAPI (Healthy cells), FITC (Necrosis cells) and Cy5 filters (Apoptotic cells)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. CytoCalcein™ Violet 450 stock solution (200X):
Add 100 µL of DMSO into the vial of CytoCalcein™ Violet 450 (Component D) to make 200X CytoCalcein™ Violet 450 stock solution. Protect from light.

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

Prepare and incubate cells with Apopxin™ Deep Red:

1. Treat cells with test compounds for a desired period of time (4-6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
   
   
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 100X Apopxin™ Deep Red (Component A) into the cells. 
   
   
5. Optional 1: Add 1 µL of 200X Nuclear Green™ DCS1 (Component C) into the cells for necrosis cells.
   
   
6. Optional 2: Then add 1 µL of 200X CytoCalcein™ Violet 450 stock solution into the cells for healthy cells staining.
   
   
7. Incubate at room temperature for 30 to 60 minutes, protected from light.
   
   
8. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.
   
   
9. Monitor the fluorescence intensity using a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel) or fluorescence microscope with DAPI (Healthy cells), FITC (Necrosis cells) and Cy5 filters (Apoptotic cells).

Analyze cells using a flow cytometer:

1. Quantify Apopxin™ Deep Red binding using a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel). Note: The flow cytometric analysis of Apopxin™ binding to 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.

 Analyze cells using a fluorescence microscope:

1. Pipette the cell suspension after incubation, rinse 1-2 times with Assay Buffer, and then resuspend the cells with Assay Buffer.
   
   
2. Add the cells on a glass slide that is covered with a glass cover-slip or a black wall/clear bottom 96-well microplate. Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip (or a black wall/clear bottom 96-well microplate). After incubation with Apopxin™ Deep Red, rinse 1-2 times with assay buffer, and then add assay buffer back to the cover-slip (or a black wall/clear bottom 96-well microplate). Invert cover-slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after the incubation with Apopxin™ Deep Red and visualized under a microscope.
   
   
3. Analyze the apoptotic cells with Apopxin™ Deep Red under a fluorescence microscope using Cy5 filter. Measure the cell viability using FITC filter when Nuclear Green™ DCS1 is added, and/or Violet filter when CytoCalcein™ Violet 450 is added into the cells. The red staining on the plasma membrane indicates the Apopxin™ Deep Red binding to PS on cell surface.