Cell Meter™ Live Cell Caspase 9 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-LEHD-FMK into cell solution at 1:150 ratio
3. Incubate at room 37^oC for 1 hour
4. Pellet the cells, wash and resuspend the cells with buffer or growth medium
5. Optional: label the cells iwth DNA stain Propidium Iodide or Hoechst 33342 
6. Analyze the cells with flow cytometer using 530/30 nm filter (FiTC channel), fluorescence microscope using FITC filter or fluorescence micro plate reader at 490/525 nm (Cutoff=515 nm)

Important notes
Thaw all the components at room temperature before use.

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

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

1. Examples for inducing apoptosis in suspension culture:
   Treat Jurkat cells with 2 µg/ml camptothecin for 3 hours
   Treat Jurkat cells with 1 µM staurosporine for 3 hours
   Treat HL-60 cells with 4 µg/ml camptothecin for 4 hours
   Treat 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 150 X FAM-LEHD-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-LEHD-FMK labeling. The appropriate incubation time depends on the individual cell type and cell concentration used.
   
   
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-LEHD-FMK is fluorescent, thus it is important to wash out any unbound reagent to eliminate the background. 
   
   
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 fluorescent microplate reader according to table 1 or table 2. For fluorescence microscopy and fluorescent microplate reader, place 100 µL of the cell suspensions into each of wells of a 96-well black microtiter plate. Note: For detached cells, the concentration of cells should be adjusted to 2 - 5 X 10⁵ cells/100 µL aliquot per microtiter plate well. 

Table 1. Fluorescence intensity monitoring for flow cytometry and fluoresence microscopes.

Table 2. Fluorescence intensity monitoring for fluorescence microplate readers.