Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 2 × 10⁶ cells/mL
2. Add TF3-DEVD-FMK into cell solution at 1:150 ratio
3. Incubate at room temperature for 1 hour
4. Pellet the cells, wash and resuspend the cells with buffer or growth medium
5. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 550/595 nm (Cutoff = 570 nm), fluorescence microscope with TRITC filter, or flow cytometer with FL1 channel

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

1. TF3-DEVD-FMK stock solution (150X):
Add 50 µL of DMSO into the vial of TF3-DEVD-FMK (Component A) to make 150X TF3-DEVD-FMK stock 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. At the same time, culture a non-induced negative control cell population at the same density as the induced population for every labeling condition.  Here are a few examples for inducing apoptosis in suspension culture:
   
   1. Treating Jurkat cells with 2 µg/ml camptothecin for 3 hours.
      
   2. Treating Jurkat cells with 1 µM staurosporine for 3 hours.
      
   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. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
      
2. Add 150X TF3-DEVD-FMK stock solution into the cell solution at a 1:150 ratio, and incubate the cells in a 37°C, 5% CO₂ incubator for 1 hour. Note: The cells can be concentrated up to ~ 5 X 10⁶ cells/mL for TF3-DEVD-FMK labeling. For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with TF3-DEVD-FMK. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
3. Spin down the cells at ~ 200g for 5 minutes, and wash cells with 1 mL Washing Buffer (Component B) twice. Resuspend the cells in desired amount of washing buffer. Note: TF3-DEVD-FMK is fluorescent, thus it is important to wash out any unbound reagent to eliminate the background. For detached cells, the concentration of cells should be adjusted to 2 - 5 X 10⁵ cells/100 µL aliquot per microtiter plate well.
   
   
4. If desired, label the cells with a DNA stain (such as Nuclear Green™ DCS1 for dead cells, or Hoechst for whole population of the cell nucleus stain).
   
   
5. Monitor the fluorescence intensity by fluorescence microscopy, flow cytometer, or fluorescence microplate reader at Ex/Em = 550/595 nm (for Nuclear Green™ DCS1, Ex/Em = 490/525 nm, for Hoechst dyes, Ex/Em = 350/461 nm).
   
   For flow cytometry: Monitor the fluorescence intensity using the channel with Ex/Em = 550/595 nm (FL1 channel for Nuclear Green™ DCS1 staining). Gate on the cells of interest, excluding debris.
   
   For fluorescence microscope: Place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Observe cells under a fluorescence microscope using TRITC channel (FITC channel for Nuclear Green™ DCS1 staining, DAPI channel for Hoechst staining).
   
   For fluorescence microplate reader: Place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Monitor the fluorescence intensity (bottom read mode) with a fluorescence microplate reader at Ex/Em = 550/595 nm (Cutoff = 570 nm). Note: If it is necessary to equilibrate the cell concentrations, adjust the suspension volume for the induced cells to approximate the cell density of the non-induced population. This adjustment step is optional if your cell treatment does not result in a dramatic loss in stimulated cell population numbers.