Aquacosmos ratio

For the assay of protease activity, an AmpliteTM Universal Fluorimetric Protease Activity Assay Kit, Green Fluorescence (AAT Bioquest, Sunnyvale, CA, USA) was used. To examine the time course of protease activity in the medium with or without SBTI (final concentration 1 μM), bivalirudin (final concentration 100 ng/ml) or tranexamic acid (final concentration 10 mM), we added 180 μl of the AmpliteTM kit substrate (fluorescent casein conjugate, diluted 1:100) to a culture of differentiated human keratinocytes in 14 mm dishes, then added 20 μl of BSS(+) solution with/without Cry j1 (100 ng/ml). The fluorescence (excitation filter of 455–485 nm and emission filter of 500–545 nm) was imaged with a high-sensitivity CCD camera (ORCA-R2, Hamamatsu Photonics, Hamamatsu, Japan) under the control of a Ca²⁺ analyzing system (AQUACOSMOS/RATIO, Hamamatsu Photonics).

Changes of intracellular calcium concentration in single cells were measured with Fura-2 AM according to the manufacturer’s instructions (Molecular Probes Inc., OR, USA). Briefly, cells were loaded with 5 μM Fura-2 AM at 37 °C for 45 min. After loading, the cells were rinsed with balanced salt solution containing (in mM): NaCl 150, KCl 5, CaCl₂ 1.8, MgCl₂ 1.2, HEPES 25, and D-glucose 10 (pH 7.4), abbreviated as BSS(+), and incubated for a further 10 min at room temperature to allow de-esterification of the loaded dye.
The coverslip was mounted on an inverted epifluorescence microscope (ECLIPSE Ti, Nikon, Tokyo, Japan), equipped with a 75 W xenon lamp and band-pass filters of 340 and 380 nm. Imaging was done with a high-sensitivity CCD camera (ORCA-R2, Hamamatsu Photonics, Hamamatsu, Japan) under the control of a Ca²⁺ analyzing system (AQUACOSMOS/RATIO, Hamamatsu Photonics). The intracellular calcium concentration was measured every second.

Changes of intracellular calcium concentration in single cells were measured with Fura-2 AM according to the manufacturer’s instructions (Molecular Probes Inc., Eugene, United States of America). Briefly, cells were loaded with 5 μM Fura-2 AM at 37°C for 45 min. After loading, the cells were rinsed with balanced salt solution containing (in mM): NaCl 150, KCl 5, CaCl₂ 1.8, MgCl₂ 1.2, HEPES 25, and D-glucose 10 (pH 7.4), abbreviated as BSS(+), and incubated for a further 10 min at room temperature to allow de-esterification of the loaded dye.
The coverslip was mounted on an inverted epifluorescence microscope (ECLIPSE Ti, Nikon, Tokyo, Japan), equipped with a 75 W xenon lamp and band-pass filters of 340 and 380 nm. Imaging was done with a high-sensitivity CCD camera (ORCA-R2, Hamamatsu Photonics, Hamamatsu, Japan) under the control of a Ca²⁺ analyzing system (AQUACOSMOS/RATIO, Hamamatsu Photonics). The intracellular calcium concentration was measured every second with or without addition of 3-phenylpropyl propionate (3PPP) (50 μM, 500 μM or 1 mM), SQ-22536 (100 μM) or L-cis-diltiazem (100 μM). Addition of 3PPP, 3PPP + SQ-22536 mixture and 3PPP + L-cis-diltiazem mixture were performed by using reflux system (VC-6 SIX CHANNEL VALVE CONTROLLER, Hamamatsu Photonics, Hamamatsu, Japan).