Benzamidine hcl

Bovine knee joints (n = 9) were obtained from the local abattoir (Atria Oyj, Kuopio, Finland) and specimens were harvested. In total, 18 samples (n = 9 + 9, osteochondral plugs, diameter = 7 mm) were prepared, two adjacent samples from lateroproximal parts of the patellae. One specimen from each pair was digested for 2.5 hours in 1 mg/mL trypsin (Sigma-Aldrich, St. Louis, MO, USA. trypsin treatment primarily affects cartilage proteoglycans, cleaving the molecules and altering the properties of the specimens; treatemen was specifically utilized to alter the relaxation times of the specimens.) at 37°C while the other sample remained in phosphate buffered saline (PBS) containing enzyme inhibitors (5 mM EDTA (VWR International LLC, West Chester, PA, USA) and 5 mM Benzamidine HCl (Sigma-Aldrich)). After the treatment, the samples were stored frozen at −20°C, acknowledging potential changes in the MRI characteristics of the specimens (29 (link),30 (link)). Prior to the measurements samples were thawed in a water bath at room temperature, and after the MRI measurement they were rinsed with fresh PBS and frozen again. A subset (n = 3 + 3) of the samples was subjected to another thaw-freeze cycle and an extra measurement; the subset was immersed in 1.0 mM gadopentetate solution for 24 hours and re-measured again using a subset of the same protocol.

Lysates were prepared from B cell pellets stored at −80°C or from B cell cultures collected by centrifugation. Approximately 5 × 10⁶ B cells were resuspended in 200 μL B cell lysis buffer (0.5% NP-40, 50 mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM Benzamidine HCl (B6506, Sigma), 1 mM PMSF (36978, Thermo Scientific), 5 μM β-mercaptoethanol, and 1 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail tablet per 10 mL buffer (11836170001,Sigma)), and incubated on ice for 30 min. Lysates were sonicated using a Branson digital sonifier with a microtip until no longer viscous, and protein concentrations were measured by Bradford assay (5000001, Bio-Rad). 4x protein sample buffer (10% SDS, 50% glycerol, 0.05% Bromophenol blue, 200 mM Tris pH 6.8, 40 mM EDTA, 20% β-mercaptoethanol) was added to a final concentration of 1x, samples were boiled at 98°C 10 min and stored at −20°C.

Twenty-six mated, osteochondral plug pairs were cored from the femoral groove (12mm diameter) and patella (7mm) of seven freshly slaughtered, skeletally mature cows. All plugs were then frozen at −20 °C in 400 mOsm/kg saline containing GIBCO Anti-Anti stock solution (5x; Invitrogen, Grand Island, NY), ethylenediaminetetraacetic acid (5 mM; Sigma, St. Louis, MO), and benzamidine HCl (5 mM; Sigma B6506, St. Louis, MO). These same additives were included in all solutions that were exposed to the samples to prevent nonspecific cartilage degradation during the experiments as a result of the samples being left at room temperature or at 4 °C for extended periods. Prior to testing, all samples were thawed overnight at 4 °C. Twelve plug pairs were used to evaluate the frictional properties and wear prevention of saline, bovine synovial fluid (BSF), and 2.8M TEG during a long-duration torsional friction test (Study 1). In Study 2, three plug pairs were used to directly compare 2.8M TEG to BSF by testing the pairs in 2.8M TEG following testing in BSF. Finally, eleven plug pairs were used to evaluate the ability of BSF, Synvisc, and 2.8M TEG to improve the frictional properties of previously worn cartilage (Study 3). Group bias was controlled by using neighboring plug pairs from the same knees for each lubricant group.