NucView 488 Caspase Detection in Living Cells

Figure 1. Staurosporine-induced MDA-MB-231 cells were first incubated with 1μM DEVD-NucView 488 caspase-3 substrate at 37oC for 30 minutes, followed by regular cell fixation in 3.7% formaldehyde. The fixed cells were then permeabilized and stained with sulforhodamine 101-phalloidin (Texas Red -phalloidin)(#00033). The nuclear DNA of the apoptotic cell was stained green by the enzymatically released DNA dye while the cell skeleton was stained red by the F actin-binding phalloidin.

Figure 2. Apoptotic Jurkat cells successively incubated with sulforhodamine 101-annexin V (Texas Red-annexin V) (#29002) and DEVD-NucView™ 488 caspase-3 substrate for 15 minutes each. The annexin V stained phosphatidylserine (PS) on the cell surface red while the substrate was cleaved by caspase-3 to release a DNA dye that stained the nuclear DNA green.

Figure 3. Flow cytometry detection of caspase-3 activity in Jurkat cells using DEVD-NucView™ 488 caspase-3 substrate following apoptotic induction with staurosporine for varying amounts of time (1h, 2.5 h and 5 h, respectively). Before each analysis, the induced cells were incubated with NucView™ 488 caspase-3 substrate (10 μM) for 15 min. Detected percentages of caspase-3-positive cells were 0% at time zero (no staurosporine induction as control) (red), 10% at 1 hour (blue), 80% at 2.5 hours (purple) and 97% at 5 hours (sky blue), respectively.

Figure 4. Titrations of NucView 488 caspase-3 substrate (1 μM) and its enzymatically cleaved product (1 μM) with dsDNA in pH 7.4 TE buffer as monitored by fluorescence at 530 nm (excitation at 475 nm) on a fluorescence microplate reader.