Intracellular cathepsin activity was detected in THP-1 cells. THP-1 cells were seeded onto chamber slides and were then treated with PMA (5 ng/mL for 48 hours) to become macrophage-like. After 48 hours, media containing PMA was replaced with fresh media, and cells were allowed to recover for 4 days, and then were exposed to the cathepsin inhibitor, CA-074 Me for 3 hours (lower row of images), or were untreated (upper row of images). Following treatment, samples were stained with R110-(RR)2 for 1 hour at 37°C and Hoechst 33342 for 15 minutes at room temperature, and then were imaged. Intracellular localization of the hydrolyzed (fluorescent) R110 product was detected using a Logos iRiS Digital Cell Imaging System equipped with a EGFP (Ex 470/30, Em 530/50) and a DAPI (Ex 375/28, Em 460/50) LED filter cubes at 20X. The images below show untreated cells (upper row of images) and inhibitor-treated cells (lower row of images) stained with R110-(RR)2 (left most images), Hoechst 33342 (middle images), or an overlay of both the EGFP and DAPI channels (rightmost images).