D5648

From the mouse: Livers were perfused through a cannulated vena cava with a prewarmed solution of sterile PBS and high glucose DMEM media containing type I collagenase solution (Thermo Fisher Scientific, Catalog # 17100017, 0.5 mg/ml) using peristaltic pump running at 3 ml/min for 10 min for each solution. Hepatocytes were released from excised livers in a 10 cm Petri dish, filtered through a 70 μm cell strainer, and pelleted at 50 g for 90 s at 4°C. Viability was determined by trypan blue staining, and cells from preparations with 85% viability or greater were plated in 6-well plates precoated with collagen I (Sigma-Aldrich, Catalog # C8919). Cultures were maintained in high glucose DMEM (Sigma-Aldrich, Catalog # D5648) containing 0.2% FBS and penicillin-streptomycin for 24 h prior treatment. From the rat: Hepatocytes were isolated from ether-anesthetized male Fisher 344 rats by in situ collagenase perfusion and cultured in culture dishes coated with 6.3 mg/ml Matrigel (BD Bioscience, Franklin Lakes, NJ) in Waymouth’s medium supplemented with insulin (0.15 μM) and penicillin/streptomycin (100U/ml) for 5 days at 37°C in 5% CO₂.

Human liver cell lines HepG2 (RRID:CVCL_0027), cultured in Eagle’s minimum essential medium (#M0643, Sigma–Aldrich), and HA22T (synonyms HA22T/VGH, RRID:CVCL_7046), cultured in DME medium (#D5648, Sigma–Aldrich) supplemented with 0.1 mM non-essential amino acids, were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Human liver cell lines Hep3B (synonyms Hep3B2.1-7, RRID:CVCL_0326) and Huh7 (RRID:CVCL_0336) were obtained from Dr. Hui-Chun Wang (Kaohsiung Medical University), and Huh6 (RRID:CVCL_4381) was obtained from Dr. Chia-Hung Yen (Kaohsiung Medical University). These cells were cultured in DME medium. Human embryonic kidney 293 T cells (synonyms HEK293T, RRID:CVCL_0063) were as previously described^{35 (link)}. All cells were maintained in medium supplemented with 10% fetal bovine serum (#26140, Thermo Fisher Scientific). All cell lines were confirmed to be mycoplasma-free and authenticated within the last three years using the short‐tandem repeats profiling method (Promega GenePrint 24 System) by Genelabs (Taipei, Taiwan).

NIH 3T3 cells (ACC 59, DMSZ, Braunschweig, Germany) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; D5648; Sigma Aldrich, Munich, BY, Germany) supplemented with 10% fetal calf serum (FCS; A15-101; PAA Cell Culture Company, Pasching, Austria) and 1% penicillin/streptomycin (P4333; Sigma Aldrich), in a humidified environment at 37 °C and 5% CO₂ (standard incubator Hera Cell 240, Heraeus Holding GmbH, Hanau, Germany). Cell culture reagents and chemicals were obtained from Sigma Aldrich unless indicated differently. Culture flasks and well plates were purchased from Sarstedt AG (Nümbrecht, Germany).

The ME49 strain of Toxoplasma gondii (ATCC^® 50611™, second haplogroup) was maintained as tachyzoites in parasite culture medium, which contains DMEM with 3% HIFBS (heat-inactivated FBS; 1 h at 56 °C). Infected cells were incubated at 37 °C and 5% CO₂. Bradyzoites were induced in vitro using the high (pH 8.2) pH shock method [50 (link)]. Bradyzoite differentiation medium contained high glucose DMEM powder (D5648, Sigma–Aldrich), 50 mM HEPES (H3375, Sigma–Aldrich), and 1% FBS. Media were adjusted to pH 8.2 with freshly made 1 M NaOH (S8045, Sigma–Aldrich) and sterilized by filtration. The medium was replaced every 1–2 days with fresh differentiation medium, which reduces the likelihood of bradyzoites reverting to tachyzoites. After 7 days, differentiated bradyzoites were confirmed with immunofluorescence assays using rabbit MAG1 antibodies as bradyzoite-specific markers and rabbit SAG1 antibodies as tachyzoite-specific markers (see Section 4.4 and Section 4.5). Six parasitized monolayers were harvested after 7 days by scraping, and bradyzoites were purified by passage through a 27 gauge needle, washed in phosphate-buffered saline (PBS) and filtered through a 0.45 μm filter. Parasites were enumerated, and pellets were resuspended in phenozol for RNA extraction using a Total RNA Mini and Clean-Up RNA Concentrator (A&A Biotechnology, Gdańsk, Poland).

Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; D5648 Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS; HyClone), 10 units/ml penicillin and 10 µg/ml streptomycin (PeSt, HyClone). The patient isolate SARS-CoV-2/01/human/2020/SWE (GenBank accession: MT093571.1) was provided by the Public Health Agency of Sweden, Stockholm. The viral stock was grown in VeroE6 cells for 48 h and titrated by a plaque assay. Briefly, VeroE6 cells (4x10⁵/well) were seeded in 12-well plates 12–24 h before infection, and a 10-fold serial dilution of virus was added. After 1 h, 2 ml semisolid overlay containing DMEM + 2% FBS + PeSt + 1.2% Avicel RC/CL was added and cells were incubated at 37 °C in 5% CO₂. After 65 h, the overlay was removed and cells were fixed with 4% formaldehyde for 30 min, washed with PBS and stained with 0.5% crystal violet in 20% MeOH for 5 min. Plates were washed with water and the plaques were counted. To perform a neutralisation assay, serum samples were heat-inactivated at 56 °C for 30 min and 10-fold dilutions of sera were prepared in virus stock (250 PFU/ml in DMEM + PeSt). The virus/sera mix was incubated at 37 °C in 5% CO₂ for 1 h, then 400 µl virus/sera inoculum were added to VeroE6 cells and the plaque assay performed as described above.

MIN6 cells were grown, according to the original protocol in Dulbecco’s modified Eagle’s medium (DMEM) (D5648, Sigma-Aldrich) [18 (link)]. Only cells in earlier passages (from 5 up to 10 times) were used in the present study except for Online Resource 2 [19 (link)]. On the day of culture, poly-l-lysine hydrobromide (PLL) (P6282, final concentration 1/500, Sigma)-coated, small glass coverslips (No. 0, Matsunami) were placed on 35 mm non-coated dish (Iwaki). Cells for the confocal measurement were seeded at a density of 1000 cells per cover slip. Culture medium was half exchanged every 3 days.