) were used at a concentration of 0.5 mg/ml. Visualization of the reaction
selleck compound product was achieved through the presence of 1 mg/ml Fast Blue B (FFB, pure, tetrazotized Di-2-anisidine ZnCl2, Serva) in the reaction mixture. 10 μm cryostat sections were pre-treated with an ice-cold mixture of acetone and chloroform (1:1) for 5 min. Slides were air-dried for 30 min at RT prior to the incubation with the substrate solution. The following substrates were used: Gly-Pro-MNA in 0.1 M PBS pH 7.0 for DPP IV, Ala-MNA in 0.1M PBS pH 7.0 for APN and Lys-Ala-MNA in 0.1 M cacodylate buffer pH 5.5 for DPP II . Incubation time was 30 min for APN and DPP IV and 60 min for DPP II at 37°C. After washing in bi-distilled water slides were mounted with Kaiser’s glycerol gelatine (Merck). Some sections were counterstained GW3965 mouse with hemalaun. For controls, the group-specific inhibitors (1 mM phenylmethanesulfonylfluoride and 1 mM diisopropylfluorophosphate, Sigma for DPP II and DPP IV and 10 mM 1,10-phenanthroline, Serva) were included in the incubation mixture. Physiological characterization of thyrocytes Cell culture Primary culture of porcine thyrocytes was performed as described previously . In brief, connective tissue was removed from thyroids of 10–20 pigs and thyroid glands were dissected into
pieces of 0.5 – 1 cm3. The pieces were incubated with 1 l 0.5% dispase mafosfamide II (Roche) in Earle’s salt solution (Gibco) for 2h at 35°C. The incubation solution was constantly stirred and aliquots of 150 ml were taken and sieved through a tea sieve. The cell suspensions were diluted 1:3 with Earle’s solution and centrifuged (200 g for 7 min at 4°C). Cells were cultured in 6-well culture plates (Falcon®) at a density of 3×106 cells/well in NCTC-135 medium supplemented with Ultroser G (3% v/v; Biosepra) and 1 μg/ml hydrocortisone and antibiotics. Human thyrocytes were also isolated from euthyroid
goiters using the same protocol. 1 mU/ml porcine TSH (Sigma-Aldrich) was added to induce the formation of follicles. Cells were also cultured in the absence of TSH. Cell number and cell viability were determined in an automatic mode based on the electrical sensing zone method (CASY Technology). For the localization of protease activities, cells (1.5×106) were seeded on cover slips placed at the bottom of 6-well plates. After 48 h of incubation, cover slips were either fixed in neutral selleck chemicals llc buffered 4% formaldehyde solution with 30% sucrose for 10 min at RT, rinsed in PBS and infiltrated for 30 min at RT in distilled water containing 30% sucrose and 1% gum arabicum or placed immediately into an ice-cold mixture of acetone and chloroform (1:1) for 5 min and then stored at −20°C until assayed for protease detection (see above). Iodide uptake For iodide uptake, 2.6 x105 cells/well were plated in 48-well plates (Costar®) and treated with either 1.