Preparation and Characterization of Fibrin Matrices

Commercial lyophilized Fb powder was dissolved in 0.9% aqueous NaCl at 37 °C and transferred to buffer 1 or buffer 2 using Zeba 7K MWCO desalting columns (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined using extinction coefficients E^0.1%_280nm for Fb: 1.51 [64 (link)], binase: 2.2 [9 (link)], trypsin: 1.6 [65 (link)]. Tb, at a concentration of 113 NIH units/mg, was dissolved in buffer 1. Stock solutions of Fb and Tb were aliquoted, frozen in liquid nitrogen and stored at −18 °C until use. Stock solutions of trypsin at a concentration of 1 mM, binase (14 mg/mL), and RG (10 mg/mL) were prepared immediately before measurements.
Fibrin coagulation was performed by the introduction of Tb, but without the addition of external calcium, in order to exclude the toxic effect of Ca²⁺ ions on the cells under study.