Cell Meter™ Multiplexing Live, Apoptotic and Necrotic Cell Detection Kit III *Triple Fluorescence Colors*

Protocol summary

1. Prepare cells with test compounds
2. Add triple fluorescence working solution (100 µL/sample)
3. Incubate at room temperature or 37^oC for 30 - 60 minutes
4. Analyze with a fluorescence microscope at FITC channel (apoptosis), DAPI channel (necrosis) or Texas Red/Cy5 channel (healthy cells)

Important notes
We treated HeLa cells with staurosporine (SS) for 4 hours at 37^oC to induce cell apoptosis. See Figure 1 for details.
Thaw all components to room temperature before beginning protocol.

Add 10 µL of 100X Annexin V-iFluor™ 488 conjugate (Component A), 5 µL of 200X Nuclear Blue™ DCS1 (Component C) and 5 µL of 200X Cellbrite™ Red (Component D) to 1 mL of Assay Buffer (Component B). The triple fluorescence assay solution is stable for at least 1 hour at room temperature. Note: As the optimal staining conditions may vary depending on different cell types, it’s recommended to determine the appropriate concentration of Component A, C and D individually.

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

1. Remove cell culture medium and test compounds after treatment.
   
   
2. Add 100 µL/well (96-well plate) or 50 µL/well (384-well plate) of triple fluorescence assay solution. Incubate at 37^oC for 30 to 60 minutes, protected from light.
   
   
3. Wash cells with HBSS, PBS or buffer of your choice twice.
   
   
4. Analyze the apoptotic cells with Annexin V-iFluor™ 488 conjugate under fluorescence microscope with a FITC filter. The green staining (Ex/Em = 494/520 nm) on the plasma membrane indicates the Annexin V-iFluor™ 488 conjugate binding to PS on cell surface. Monitor the fluorescence intensity with a DAPI filter (Ex/Em = 350/461 nm) for necrosis, Texas Red or Cy5 filter (Ex/Em = 613/631 nm) for live cells using a fluorescence microscope (See Figure 1 for details).