Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare cells 
2. Remove the growth medium, wash cells
3. Incubate Caspase 3/7 Substrate working solution at  37 ºC for 2 hours 
4. Wash cells and replace with growth medium
5. Induce apoptosis
6. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 405/570 nm (Cutoff = 540 nm), fluorescence microscope with DAPI filter, or flow cytometer with Ex/Em = 405/470 nm filters

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

Add 2 μL of 500X ApoBrite™ V570 Caspase 3/7 Substrate stock solution (Component A) into 1 mL HHBS (Component B) to make 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution. 

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

1. Culture cells to a density optimal for apoptosis induction according to your specific induction protocol, but not to exceed 2 x 10⁶ cells/mL for suspension cells. For adherent cells, plate cells at 30,000 - 80,000 cells/well/96-well plate overnight.
        
2. Remove growth medium, and wash once with HHBS (Component B) or buffer of your choice.
   
   
3. Add 100 µL/well/96 well plate of 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution, and incubate at 37 ºC, 5% CO2 incubator for 2 hours.
   
   
4. Remove 1X ApoBrite™ V570 Caspase 3/7 Substrate working solution and wash once with HHBS (Component B).
   
   
5. Add growth medium back to the ApoBrite™ V570 Caspase 3/7 loaded cells, and treat the cells as desired for apoptosis. Here are a few examples for  inducing apoptosis in cell culture:
   
   
   1. Treating Jurkat cells with 2 µg/ml camptothecin for 3 hours.
      
      
   2. Treating Jurkat cells with 1 µM staurosporine for 3 hours. Hela cells for 1 hour.
      
      
   3. Treating HL-60 cells with 4 µg/ml camptothecin for 4 hours.
      
      
   4. Treating HL-60 cells with 1 µM staurosporine for 4 hours.
      
      
   5. Treating Hela cells with 1 µM staurosporine for 1 hour. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
      
      
6. If desired, label the cells with a DNA stain (such as 7-AAD for dead cells).
   
   
7. Monitor the fluorescence intensity by fluorescence microscopy, flow cytometer, or fluorescence microplate reader at Ex/Em = 405/570 nm (for 7-AAD, Ex/Em = 535/650 nm).
   
   For flow cytometry (suspension cells): Monitor the fluorescence intensity using Ex/Em = 405/570 nm filters (FL3 channel for 7-AAD staining). Gate on the cells of interest, excluding debris.
   
   For fluorescence microscope:  Observe cells under a fluorescence microscope using Ex/Em = 405/570 nm or DAPI channel (TRITC channel for 7-AAD staining).
   
   For fluorescence microplate reader: Monitor the fluorescence intensity (bottom read mode) with a fluorescence plate reader at  Ex/Em = 405/570 nm (Cutoff = 540 nm).