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

Protocol summary

1. Prepare cells with test compounds (200 µL/sample)
2. Add Apopxin™ Green assay solution
3. Incubate at room temperature for 30 - 60 minutes
4. Analyze cells with a flow cytometer with emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic 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™ Green:

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™ Green (Component A) into the cells. 
   
   
5. Optional 1: Add 1 µL of 200X 7-AAD (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 emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic cells).

Analyze cells using a flow cytometer:

1. Quantify Apopxin™ Green binding using a flow cytometer with emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic cells). 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™ Green, 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™ Green and visualized under a microscope.
   
   
3. Analyze the apoptotic cells with Apopxin™ Green under a fluorescence microscope using the FITC filter. Measure the cell viability using Texas Red filter when 7-AAD is added, and/or DAPI or Violet filter when CytoCalcein™ Violet 450 is added into the cells. The green staining on the plasma membrane indicates the Apopxin™ Green binding to PS on cell surface.