Trypsin 9

Mesentery was removed and enzymatically dissociated in 20–30 mL of culture medium (alpha-MEM, pH 7.4) containing collagenase 1 A, trypsin IX and papain 2x crystallized (Sigma-Aldrich, USA) at 37 °C in 5% CO₂ atmosphere for 2 hours with gentle agitation. At the end of the incubation, the cells recovered by centrifugation (5 min, 150 g). Each pellet was submitted to annexin V-FITC and propidium iodide (PI) staining and flow cytometry analysis. Living cells have double-negative Annexin-V⁻PI⁻ phenotype while dead cells are positive to Annexin-V and/or PI. Results were representative of three independent experiments.

The MA104 cell line (American Type Culture Collection, DC, USA) was maintained in minimum essential medium (MEM; Sigma, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and antibiotics and antimycotic (Lonza, Basel, Switzerland). Rhesus rotavirus (RRV) strain was kindly provided by Harry Greenberg (Stanford, CA, USA), and human rotavirus (HRV) strain ST-3 genotype G4P [6 (link)] was kindly provided by David Allen (Public Health England, UK). Working stocks were obtained by infecting MA104 cells at a multiplicity of infection (MOI) of 0.1 in MEM supplemented with 0.5 μg/mL trypsin IX (Sigma), and harvesting at 100% cytopathic effect.