Figure 5

A. Caspase-3 Assay: Jurkat cells were treated with 10 μM AMD4 or AMD5 for the early time-points of 3 hrs and 6 hrs. Following treatment, Jurkat cells were incubated with DEVD-AFC peptide to detect caspase-3 activity; fluorescence was measured at excitation 400 nm and emission 505 nm. Activation of caspase-3 is a common event in cells undergoing apoptosis. The level of caspase-3 activity was calculated per microgram of protein and then displayed as a percent increase in fluorescence from caspase-3 activity in untreated cells (control). Values that are statistically significant to p < 0.05 are indicated with an asterisk. B. Mito-Casp Assay: Jurkat cells were treated with 10 μM AMD5 for 48 hrs. Following treatment, Jurkat cells were incubated with a mitochondrial membrane potential (MMP) sensitive dye, and fluorescence was measured at excitation 574 nm and emission at 595 nm. Loss of MMP indicates permeable mitochondria which allows for numerous cytotoxic materials to leak into the cytosol, ending in apoptosis. Loss of MMP is represented as a drop in relative fluorescence units per 10,000 cells. Values that are statistically significant to p < 0.05 are indicated with an asterisk.