Biokinetics reader

Kal activity was spectrophotometrically monitored via conversion of the chromogenic substrate H-D-Pro–Phe–Arg-pNA·2HCl, S-2302 (0.5 mM; HYPHEN BioMed, Neuville-sur-Oise, France) using a Bio-Kinetics Reader (BioTek Instruments, Inc., Winooski, VT, USA), as described previously (28 (link), 29 (link)). To determine the specific effect of DSS on the activation of Kal, a monoclonal antibody (IgG1) against Kal M0202-H03 (Dyax, now a part of Shire, Burlington, MA, USA) was used, and an isotype-matched IgG1 (Sigma) was used as a control. Human and mouse plasma was prepared using sodium citrate as coagulant. In brief, 90 µL of diluted plasma (1:1) was preincubated with 10 µL of antibody or control buffer for 30 min. Ninety microliters of pretreated plasma were incubated with 10 µL of DSS for 10 min. Ten microliters of DSS-treated plasma were added to 90 µL of substrate in 96-well plate. A chromogenic substrate of FXIIa (SPECTROZYME^®, Sekisui Diagnostics, Lexington, MA, USA) was used to measure FXIIa generation. The high sensitivity and specificity for FXIIa was demonstrated in a previous study (30 (link)). The positive control for FXII activation was 100 µg/mL of kaolin. The optical density of 0.5 mM substrate hydrolysis was measured at 405 nm using a spectrophotometer (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) (31 (link)).

After culturing for 1, 3, 5, and 7 days, cell proliferation was determined using the MTT method. In brief, each well was aspirated and washed with PBS three times. Next, 700 μL of DMEM (with 10% FBS) and 70 μL of MTT (0.5 mg/mL of water) solution was added into each well and incubated at 37°C at 5% CO₂ for 3 h in the dark. The medium was then aspirated and 700 μL of DMSO was added into each well to dissolve the formazan. After shaking gently on a shaker in the dark for 15 min, the absorbance at 450 nm was measured using a microplate reader (biokinetics reader, Bio-Tek Instruments, USA). Cells grown on the culture plates only were used as the control group. The measurements were performed in triplicate for each group.