Trypsin type 9

All animal handling was performed in accordance with the Helsinki convention. One-year-old wild-type zebrafish were used in the experiments.
Ventricular cardiomyocytes were obtained from the heart by enzymatic dissociation. The fish were killed by decapitation. The heart was rapidly excised. A cannula, blunted syringe needle 32 gauge, was introduced through the aortic bulb of the isolated heart for retrograde perfusion for 10–15 min with a Ca²⁺-free solution of the following composition (in mM): 100 NaCl, 10 KCl, 1.2 KH₂PO₄, 4 MgSO₄, 10 HEPES, 50 taurine, 20 glucose, pH 6.9 (adjusted with KOH at room temperature). Then the heart was perfused for 25–30 min with the same solution containing proteolytic enzymes: 0.7 mg/mL collagenase type IA (Sigma-Aldrich, St. Louis, MO, USA); 0.6 mg/mL trypsin, type IX (Sigma-Aldrich, St. Louis, MO, USA), and 1 mg/mL bovine serum albumin (DIA-M, Moscow, Russia). All perfusion was carried out at room temperature, trypsin was used only to obtain cells for registration of Na⁺ current. After the end of perfusion, the atrium was removed and ventricular myocardium was destroyed mechanically (by cutting with surgical scissors and pipetting) to isolate individual cells. Cardiomyocytes were stored in the Ca²⁺-free solution at +4 °C for no more than 8 h.

The MA104 cell line was obtained from ATCC (CRL-2378.1^TM) and cultured in complete M199 media (Sigma-Aldrich) supplemented with heat-inactivated 10% fetal calf serum (FCS; heated to 56 °C for 30 min), 1% L-glutamine, and pen/strep (Corning). Incomplete M199 media was only supplemented with 1% L-glutamine and pen/strep without FCS. Cells were passaged using 0.05% 1X trypsin-EDTA (Gibco, Grand Island, NY, USA). Unless specified otherwise, all culturing and incubating conditions were performed at 37 °C and 5% CO₂; 200 µL of murine ETD rotavirus, a tissue-culture adapted EDIM strain [46 (link)], was diluted in incomplete M199 media for an MOI of approximately 0.01. The virus was activated by incubating for 20 min with 2.5 µg/mL of trypsin type IX (Sigma-Aldrich). Confluent MA104 cells were washed in incomplete M199 media. Activated virus was diluted 1 to 5 with incomplete M199 media and incubated for 50 min with the cells. Fifteen mL of incomplete M199 media and 0.5 µg of trypsin/mL were added to the flask, and the infected cells were incubated for 3–4 days until complete cell death. The virus was aliquoted and stored at −80 °C.