Stericup vacuum filtration system

Wild-type mouse ESCs (mESCs) were derived freshly (mixed 129/B6, XY) and cultured on gelatin-coated cell culture plates under naive conditions (2i/leukemia inhibitory factor (LIF)), in accordance with the approved protocol by the laboratory animal management and ethics committee of the EMBL under license 20191001MBJH. Routine passaging was performed in N2B27 basal culture medium (NDIFF, Takara, y40002), supplemented with 1 μM PD0325901 and 3 μM CHIR99021 (both from Axon Medchem), 1,000 U ml -1 LIF (in-house production), 1% FBS (Millipore) and 1% penicillin-streptomycin (Gibco). All culture media were filtered through a 0.22-μm pore Stericup vacuum filtration system (Millipore). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO 2 and were passaged every 2 d by dissociation with TrypLE (Thermo Fisher Scientific). Culture medium was replaced with fresh stocks daily. Mycoplasma contamination was tested routinely by the ultrasensitive qPCR assay (Eurofins).

Fermentative vigour without and with sulphites were measured according to Caridi et al. (2002) : flasks containing 100 ml of sterile white must (20°Brix, pH 3.20, filtered by Stericup vacuum filtration system, Millipore, Billerica, MA, USA), with and without SO 2 (100 mg l À1 ) and covered with 10 ml of sterile liquid paraffin to prevent evaporation, were inoculated in triplicate with 5 ml of 48 h precultures of each isolated yeast and incubated at 25°C. Fermentative vigour was measured as weight loss caused by CO 2 production (g CO 2 per 100 ml) after 2 and 7 days; S. cerevisiae L404 (DIPROVAL collection of the University of Bologna, Italy) was used as a positive control, not-inoculated must as negative control. Reducing sugar, ethanol, glycerol and volatile acidity were measured in must prepared as described above (without SO 2 ) using a WineScan TM apparatus (FOSS, Hilleroed, Denmark) after 15 days of fermentation. We estimated H 2 S production on BiGGY agar (Oxoid) recording the biomass colour after 48 h at 25°C (Nickerson 1953 ). bglucosidase production was assayed as in Strauss et al. (2001) onto selective medium containing 6Á7 g l À1 Yeast Nitrogen Base (Difco, Detroit, MI, USA), 5 g l À1 arbutin (Sigma-Aldrich, Saint Louis, MO, USA), 0Á2 g l À1 ammonium ferric citrate and 20 g l À1 agar (pH 5Á0).