Trypsin inhibitor from glycine max

Islets of Langerhans were isolated from euthanized adult male Sprague Dawley rats by retrograde perfusion of the pancreas^{79 (link)} with 0.5 mg/ml collagenase from Clostridium Histolyticum (Sigma, St. Louis, MO) in HBSS (Gibco) supplemented with 5 mM HEPES. The excised pancreas was transferred to HBSS containing 5 mM HEPES, 5 mM glycine and 0.165 mg/ml trypsin inhibitor from Glycine max (Sigma) and placed in 37 °C water bath until tissue was readily dissociated when shaken by hand. Digestion was terminated by addition of ice-cold HBSS with 0.4% BSA. Tissue was passed through a size 40 sieve and washed 3 times by centrifugation at 200 g for 10 s followed by gradient centrifugation in Histopaque®-1077 at 1000 g for 25 min. The islets were washed twice and handpicked under a stereomicroscope. Islets where cultured for 18 h in RPMI1640 medium containing 11 mM glucose, 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere at 37 °C with 5% CO₂ in air. 11–30 islets were transferred to 300 μl KRBH in low glucose for 2 h prior to addition of the indicated glucose concentrations alone or in the presence of BzATP (10 μM) or AZ10606120 (10 μM). Insulin release was taken as the difference between insulin in the supernatant obtained before addition of test compounds and 30 min after addition. Samples were handled as described above.

Manual microdissection of distal convoluted tubules was conducted according to a previously published protocol [47 (link)]. After perfusion of anesthetized mice with MEM media (GibcoBRL, Ref number 11058021) via left ventricle, the kidney was sagittally cut into two halves and the medulla was excised. Cortex was sliced into 1-mm thin sections that were then incubated in a digestion solution containing 4 ml MEM, 5 mM glycine, 6 mg/ml trypsin inhibitor (Trypsin inhibitor from Glycine max, Sigma-Aldrich, T9128), and 250 μg/ml collagenase (Collagenase from Clostridium histolyticum, Sigma-Aldrich, C9891), pH 7.4 at 37 °C for 25 min without shaking. The digested cortex sections were subsequently washed with ice-cold HEPES solution (NaCl 125 mM, KCl 3 mM, MgSO₄ 1.2 mM, CaCl₂ 1 mM, KH₂PO₄ 2 mM, glucose 5 mM, HEPES 32.2 mM), and the renal DCTs were manually microdissected under a stereomicroscope based on their distinct morphological characteristic.

A/WSN/33-based VLPs harboring β-lactamase-M1 (BlaM1) fusion proteins were produced essentially as described by Tscherne & Garcìa-Sastre^{42 (link)}. Briefly, HEK 293 T cells seeded onto poly-L-lysine-coated (Sigma-Aldrich) 6-well plates were transfected in Opti-MEM (Life Technologies) with 2 μg BlaM1, 500 ng pCAGGS-WSN-HA, 500 ng pCAGGS-WSN-NA per well using ViaFect (Promega) as the transfection reagent (3 µl ViaFect/µg DNA). Medium was exchanged 6 h post-transfection with Opti-MEM (Life Technologies) containing 1% penicillin-streptomycin. VLPs were collected 72 h after transfection and treated with 5 μg/mL TPCK-trypsin (Sigma-Aldrich) for efficient HA cleavage. Trypsin was inactivated with 10 μg/mL trypsin inhibitor from Glycine max (Sigma-Aldrich). Prior to infection, MDCK cells were treated with the indicated concentrations of the GRK2 inhibitor for 1 h. For infection of MDCK cells in 24-well plates, 200 μl BlaM1 VLPs were added to the cells in the presence of the compound and incubated for 4 h at 37 °C. Cells were harvested by trypsinization and incubated with the florigenic substrate CCF2-AM (ThermoFisher Scientific). Cells were analyzed on a FACSVerse System (BD) and dead cells excluded by a live/dead staining (LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, ThermoFisher Scientific).