Cellulose acetate filter

Lentivirus was generated through transfection of HEK 293 T Cells with packaging plasmids (pMD2.G and psPAX2 vectors) and previously described plasmids containing mouse wild type Ripk3 on a modified lentiviral tet-on pTRIPZ/Puro vector using the TransIT^®-Lenti transfection reagent (Mirus Bio, Madison, WI, USA) (3 (link)). After 48 hours, the culture supernatant was filtered with 0.45 μm cellulose acetate filters (VWR, Radnor, PA, USA) to collect RIPK3-encoding lentivirus. LLC-OVA cells were incubated with lentivirus in complete DMEM containing polybrene (8 μg/mL) for an additional 48 hours. Transduced cells were selected with 2 μg/ml puromycin.
For generating RIPK1, CASP8 and MLKL-deficient cell lines, the following guide RNA (gRNA) sequences were cloned into LentiCRISPRv2-Blast lentiviral vector [a gift from Mohan Babu; Addgene plasmid # 83480]: 5’- CAGACTGAGACACAGTCGAG-3’ (murine Ripk1 gRNA #1), 5’- TGTGAAAGTCACGATCAACG-3’ (murine Ripk1 gRNA #2), 5’-AGACGACACCCTTGTCACCG-3’ (murine Casp8 gRNA #1), 5’- AGATGTCAGGTCATAGATGG-3’ (murine Casp8 gRNA #2), 5’-CAAAGTATTCAACAACCCCC-3’ (murine Mlkl gRNA #1), 5’- AGGAACATCTTGGACCTCCG-3’ (murine Mlkl gRNA #2). Constructs were transduced into LLC OVA cells as described above and selected in Blasticidin (8 μg/mL).

All experiments were performed in duplicates. Shown is the arithmetic mean of the duplicates. Error bars and ± values indicate deviation from the mean.
When using CaCO₃ as buffer, 1 mL of culture broth was taken for OD₆₀₀ determination and HPLC analysis. The CaCO₃ was dissolved with HCl prior to further measurements. OD₆₀₀ was determined in an Ultrospec 10 cell density meter (Amersham Biosciences, UK), samples were diluted to an OD₆₀₀ between 0.1 and 0.8.
For HPLC analysis, centrifuged samples (13.000 g, 5 min) were filtered through cellulose acetate filters (diameter 0.2 µm, VWR, Germany) and subsequently diluted 1:10 with distilled water. Glycerol and organic acids were analyzed on a Dionex Ultimate 3000 HPLC (Dionex, USA) with an Organic Acid Resin column (CS–Chromatographie, Germany) kept at 75 °C, with a constant flow rate of 0.8 mL min⁻¹ of 5 mM sulfuric acid as eluent. For detection, a Shodex RI 101 detector at 35 °C and a variable wavelength UV detector (Dionex, USA) at 210 nm were used.
Ammonium concentration was determined by a colorimetric assay according to Willis [38 (link)].

Biogenic amines were determined by ultra-high performance liquid chromatography (UHPLC). In broth they were quantified directly from a 100 μl sample, as described by Ladero et al. (2015) (link). The BA in cheese samples were first extracted and quantified as previously described (Herrero-Fresno et al., 2012 (link)) with some modifications. Briefly, 1 g of cheese was mixed with 10 mL of 0.1 M HCl containing 0.2% (w/v) 3,3′thiodipropionic acid using an Ultra Turrax T50 homogeniser (OMNI International, United States) for 2 min at 20,000 rpm. The samples were then disrupted for 30 min in an ultrasonic bath and centrifuged at 5000 ×g for 30 min. After removing the fat layer, the supernatant was filtered through 0.45 μm cellulose acetate filters (VWR, Spain). The filtrates were deproteinised using ultra-filtration inserts (Amicon Ultracel-3K, Millipore) during centrifugation at 3500 g for about 1 h in a 5810 Eppendorf benchtop centrifuge (Eppendorf, Spain). Supernatant samples (100 μL) were then derivatised with diethyl ethoxymethylenemalonate as described by Redruello et al. (2013) (link), and the BA separated out and quantified in an H-Class Acquity UPLC^TM UHPLC system (Waters, United States) running Empower 2 software (Waters), using the conditions described in the latter paper.

Mycelium for amino acid analysis was harvested during steady-state according to the method described by de Jonge et al.^{65 (link)}. In brief, samples of 10 ml or less were quenched directly into −20 °C 40% methanol (20 ml), weighted and filtered using a vacuum pump followed by a single washing 1x with the same volume of ice-cold 40% methanol before freezing in liquid nitrogen and storage at −80 °C until extraction. For extraction, filter papers with frozen sample were directly placed in 50 ml falcon tubes containing 20 ml 73 °C hot 75% ethanol, shaken vigorously, boiled for 3 min at 95 °C, chilled on ice for 5 min, centrifuged for 5 min at 4000 × g⁻¹ and filtered over a 0.2 µm cellulose acetate filter (VWR). 1 ml aliquots were concentrated in a speed-vac (Eppendorf) for 45 min at 30 °C, centrifuged for 10 min at 10.000 × g⁻¹. Supernatant was stored at −80 °C if not used immediately for LC-MS analysis. All extractions were performed in quadruplicate per bioreactor run and analyzed in technical duplicate on LC-MS. Amino acid retention times were verified by a standard mixture (AAS18 Analytical standard; Sigma Aldrich) or dilutions of pure amino acids in 10 mM HCl (for Asn, Gln, Trp). Peak areas were corrected for extracted biomass and concentrations were calculated using a calibration curve.