AEBSF hydrochloride (Pefabloc SC) is an irreversible inhibitor of serine proteases, such as chymotrypsin, kallikrein, thrombin, plasmin, and trypsin.

The HeLa cells suspended in RPMI-1640 media containing 10% FCS are plated into each well of a 96-well microplate (5×103 cells/200 μL/well). After incubation for 24 h at 37°C, cells are treated with different doses of AEBSF (0, 25, 50, 100 μg/mL) for 48 h. Then, 20 μL fresh 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) reagent (5 μg/μL) is added into each well, and cells are cultured at 37°C in 5% CO2 for another 4 h. The media are discarded carefully, and 150 μL DMSO is added. Absorbance is read on a microplate reader at dual wavelengths of 540 and 620 nm.