Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Add 1 µL of 500X TF5-VAD-FMK into 0.5 mL of cell solution
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 1 - 4 hours
4. Pellet the cells and resuspend the cells in 0.5 mL of assay buffer or growth medium
5. Analyze cells using flow cytometer with 660/20 nm filter (APC channel)

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

1. For each sample, prepare cells in 0.5 mL warm medium or buffer of your choice at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
   
   
2. Treat cells with test compounds for a desired period of time to induce apoptosis, and create positive and negative controls.
   
   
3. Add 1 µL of 500X TF5-VAD-FMK (Component A) into the treated cells.
   
   
4. Incubate the cells in a 37°C, 5% CO₂ incubator for 1 - 4 hours. Note: 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 TF5-VAD-FMK. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
5. Wash and spin the cells twice. Resuspend the cells in 0.5 mL of Assay Buffer (Component B) or growth medium. Note: TF5-VAD-FMK is fluorescent; therefore it is important to wash out any unbound reagent to remove the background.
   
   
6. If desired, label the cells with a DNA stain (such as Green-DCS for dead cells, which can be detected with 530/30 nm filter (FITC channel)
   
   
7. If desired, fix cells.
   
   
8. Monitor the fluorescence intensity using a flow cytometer with 660/20 nm filter (APC channel). Gate on the cells of interest, excluding debris.