Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors*

1. Prepare cells with test compounds at a density of 2 × 10⁶ cells/mL
2. Add TF3-DEVD-FMK at 1:150 ratio and/or Annexin V-iFluor 488™ into cell solution at 1:100 ratio
3. Incubate the cells in a 37°C, 5% CO₂ incubator 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) and/or 550/595 nm (Cutoff = 570 nm), fluorescence microscope with FITC and TRITC filters, or flow cytometer with FL1 and FL2 channels for Annexin V-iFluor 488™ and TF3-DEVD-FMK respectively

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

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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 75 µL of DMSO into the vial of TF3-DEVD-FMK (Component A) to make 150X TF3-DEVD-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 (or not to exceed 3 x 10⁵ cells/100 µL/well in a 96-well black clear-bottom plate). 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:
2. 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.
3. Add 150X TF3-DEVD-FMK stock solution at a 1:150 ratio and/or Annexin V-iFluor 488™ (Component B) at 1:100 ratio into each well.
4. 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. Annexin V flow cytometric analysis on adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.
5. If desired, label the cells with a DNA stain (such as Hoechst for whole population of the cell nucleus stain, or propidium iodide for dead cells if the cells label with Annexin V-iFluor 488™ only).
6. Spin down the cells at ~200g for 2 minutes and wash cells with 1 mL (or 200 µL/well if using 96-well plate) Washing Buffer (Component E) twice. Resuspend the cells in desired amount of washing buffer.
   
   Note         TF3-DEVD-FMK and Annexin V-iFluor 488™ are 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.
7. Monitor the fluorescence intensity by fluorescence microscope, flow cytometer, or fluorescence microplate reader at Ex/Em = 550/595 nm for TF3-DEVD-FMK, 490/525 for Annexin V-iFluor 488™, 350/461 nm for Hoechst stain, and 535/635 for propidium iodide.

For flow cytometry: Monitor the fluorescence intensity using FL1 channel for Annexin V-iFluor 488™, FL2 channel for TF3-DEVD-FMK. 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 for TF3-DEVD-FMK, and/or FITC channel for Annexin V-iFluor 488™ (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 microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm) for Annexin V-iFluor 488™, and/or 550/595 nm (Cutoff = 570 nn) for TF3-DEVD-FMK.
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.