Trypsin type xi

Hippocampal cultures were obtained as described (Piedras-Rentería et al., 2004 (link)). In brief, whole hippocampi were dissected from newborn rats in Hanks’ Balanced Salts Solution (HBSS) (Sigma, St. Louis, MO) supplemented with 20% of Fetal Bovine Serum (Cellgro, Herndon, VA) (20% FBS-HBSS). Hippocampi were washed with 20% FBS-HBSS and HBSS and incubated for 10 min at 37°C with trypsin type XI (3.4 mg/ml) (Sigma, St. Louis, MO) plus DNAse type I (600 U/ml) (Calbiochem, Billerica, MA). After digestion, the tissue was washed with 20% FBS-HBSS and HBSS and mechanically dissociated in HBSS supplemented with 600 U/ml DNAse I and 12 mM MgSO₄. Cells were plated onto matrigel-coated coverslips (12 mm, Carolina Biological Supply, NC) at a density of 25,000–35,000 and kept in a 5% CO₂ humidified atmosphere at 37°C. The media was supplemented with thymidine -β-D-arabinofuranoside (4 μM) after the second day in culture (2 DIV, days in vitro).

Cell aggregation assays were performed as previously described (30 (link)). In brief, the cells were incubated for 10 min at 37°C in HEPES-buffered saline containing 0.01% trypsin (type XI; Sigma-Aldrich) and 2 mM CaCl₂ or 1 mM EGTA. After the addition of soybean trypsin inhibitor (Sigma-Aldrich), the cells were washed, resuspended and incubated for 30 min at 37°C with constant rotation at 70 rpm. The extent of cell aggregation was represented by the index: (Nc-Np)/Nc, where Np and Nc are the total number of particles and cells/dish, respectively.

Primary cultures of immature cortical neurons were prepared from embryonic day 19 (E19) Sprague-Dawley rats or E17 C57BL/6 mice (wild-type and DKO mutant mice) were prepared as previously described [21 (link)]. In brief, after dissection, cortices were incubated for 10 min with 10 mg/ml trypsin type XI (Sigma-Aldrich) plus 0.5 mg/ml DNase I type IV (Sigma-Aldrich) at room temperature and mechanically dissociated in Hanks’ solution, pH 7.4 (Sigma-Aldrich), with 0.5 mg/ml DNase I type IV and 12 mM MgSO₄. Dissociated neurons were transfected by electroporation using Nucleofector (Amaxa Biosystems) and plated onto either poly-L-lysine-coated 12 mm coverslips (BD Biosciences), poly-D-lysine-coated glass-bottom dishes (MatTek), or six-well dishes (BD Biosciences), and cultivated in minimum essential medium (Invitrogen) containing 5 g/L glucose, 0.2 g/L NaHCO₃, 0.1 g/L transferrin (Calbiochem), 2 mM GlutaMAX-I (Invitrogen), 25 μg/ml insulin (Sigma-Aldrich), B-27 supplement (Invitrogen), and 10 % fetal bovine serum. All primary cortical cultures were incubated in 5 % CO₂ at 37 °C.