Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 2 × 10⁶ cells/mL
2. Add FAM-LETD-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 = 490/525 nm (Cutoff = 515 nm), fluorescence microscope with FITC filter, or flow cytometer with FL1 channel

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

1. FAM-LETD-FMK stock solution (150X):
Add 50 µL of DMSO into the vial of FAM-LETD-FMK (Component A) to make 150X FAM-LETD-FMK stock solution.

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 FAM-LETD-FMK 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 FAM-LETD-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 FAM-LETD-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: FAM-LETD-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 propidium iodide 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 = 490/525 nm (for propidium iodide, Ex/Em = 535/635 nm; for Hoechst dyes, Ex/Em = 350/461 nm).
   
   For flow cytometry: Monitor the fluorescence intensity using the FL1 channel (FL2 channel for propidium iodide 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 FITC channel (TRITC channel for propidium iodide 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 plate reader at  Ex/Em = 490/525 nm (Cutoff = 515 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.

Table 1. Spectral information for measuring fluorescence intensity.