Fetal bovine serum superior

Lung fibroblasts were prepared from whole lung tissue and dermal fibroblasts were prepared from tail and ear tissue using Collagenase Type 2 (Worthington, 44N15307B). Fibroblasts were maintained in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with Penicillin-Streptomycin, l-Glutamine, Sodium Pyruvate, HEPES Buffer Solution (1% each, all Gibco) and 10% fetal serum (PAN Biotech). BMDMs were isolated from mice according to standard procedures and maintained in similar medium as fibroblasts, but with 10% fetal bovine serum superior (Biochrom) and 20 ng ml⁻¹ M-CSF (Immunotools, 12343118). BMDMs were differentiated for 6 days before plating for experiments.

COS-7 cells (kindly provided by Dr M. Schrader (University of Exeter, UK)) were cultured at 37°C in a humidified 5% CO₂ incubator in minimum essential medium Eagle α (Lonza) supplemented with 10% (v/v) fetal bovine serum superior (Biochrom AG), 2 mM Ultraglutamine-1 (Lonza) and 0.2% (v/v) Mycozap (Lonza). Cells were transfected using Invitrogen's Neon Transfection System (1050 V, 30 ms pulse width, 1 pulse). Samples for immunofluorescence microscopy were fixed and processed as described before [80 (link)]. The rabbit polyclonal antiserum against human PEX14 has been described elsewhere [81 (link)]. DAPI (Roche), the mouse anti-HA (Sigma) and anti-FLAG (Stratagene) antibodies, and the Alexa Fluor 488- (Invitrogen) or Texas Red- (Calbiochem) conjugated secondary antibodies were commercially obtained. Fluorescence was evaluated on a motorized inverted IX-81 microscope (Olympus) controlled by Cell-M software (Olympus). The technical specifications of the objectives, excitation and emission filters, and digital camera have been described elsewhere [82 (link)]. Fluorescence intensity versus distance plots (line scans) were generated using ImageJ software.

Echinococcus multilocularis metacestodes were isolated, separated from host contaminants and axenically cultivated as previously described (6 (link)). For the collection of E/S products, metacestode vesicles were kept under axenic conditions for 10 days, washed three times in 1 x PBS and resuspended in DMEM10redox i.e., DMEM ⁺ GlutamaxTM, GIBCO supplemented with 10% Fetal Bovine Serum Superior (Biochrom AG), 100 μg/ml penicillin/streptomycin (PenStrep solution, Biochrom AG), 20 μg/ml Levofloxacin (Tavanic, Sanofi-Aventis), β-mercapthoethanol (143 μM, Sigma-Aldrich, cat. M6250), 10 μM Bathocuproine disulfonic acid (Sigma, cat. B-1125) and 100 μM L-Cysteine (Sigma, cat. C-1276) under axenic conditions [see (6 (link)) for description of conditions]. After 48 h of culture, the supernatants containing the MVE/S were collected and filtered through a 0.2 μm sieve (Filtropur S filter, SARSTEDT). The total amount of E/S product proteins was determined as previously defined (6 (link)) and the E/S products stored at −80°C until use.

Human epithelial lung carcinoma cells (A549), African green monkey kidney cells (Vero) and human neuroblastoma cells (SH-SY5Y) were grown in Dulbecco’s Modified Eagle’s medium (DMEM) High Glucose (BioWest, Nuaillé, France) supplemented with 10% Fetal Bovine Serum Superior (Biochrom GmbH, Berlin, Germany), 2 mM L-Glutamine (Biochrom GmbH, Berlin, Germany), 100 U/mL penicillin and 100 μg/mL streptomycin (Paul-Ehrlich-Institut facilities, Langen, Germany) in a humidified incubator at 37 °C with 5% CO₂. Passaging of adherent cells was performed by trypsinization three times a week.

M. bovis BCG (DSMZ No. DSM 43990) was grown as previously described (Sharbati-Tehrani et al., 2004 (link)). For microscopy, BCG was fluorescently labeled with 5-(6)-carboxyfluorescein-succinylester (Sigma Aldrich) following an earlier described protocol (Agerer et al., 2004 (link)). The human acute monocytic leukemia cell line THP-1 (DSMZ ACC 16) was cultured in suspension using RPMI 1640 (Biochrom AG) supplemented with 10% fetal bovine serum superior (Biochrom AG) and gentamycin (zur Bruegge et al., 2014 (link)). Infection experiments were performed as described previously (Pawar et al., 2016 (link)).

The sera were mixed in dilution series (1:20 to 1:320) with 200 pfu of ZIKV (Asian or African strain) or WNV for 1–2 h at 37 °C. 50–100 µL of the mixture was used to infect 3 × 10⁵ Vero cells (in case of ZIKV) and 3 × 10⁵ Vero E6 cells (in case of WNV) seeded in 6-well plates for 1–2 h at 37 °C. The medium was then removed and the cells were layered with 0.4% agarose solution (seaPlaque Agarose, Lonza, Basel, Switzerland). Three to six days later, the agarose layer was removed and the cells were fixed with formaldehyde solution (4% in PBS). The plaques were visualized by staining for 15 min at RT with crystal violet solution (0.1% in 20% ethanol, Merck, Darmstadt, Germany), followed by washing with distilled water. The plates were dried and the plaques were counted. As positive controls the neutralizing antibodies 4G2 and Z004-HRP (Absolute antibody) were used. In some experiments (indicated in the figure legends), the sera and the FBS, which was used to prepare the medium, were heat-inactivated for 30 min at 56 °C. In these experiments, DMEM high glucose medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum superior (Biochrom GmbH, Berlin, Germany), 2 mM l-glutamin (Merck, Darmstadt, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin was used.

Osteoblasts were generated from long bones of mice as described previously (Rapp et al., 2018 (link)). Following digestion with 300 U/ml collagenase type IV (Sigma Aldrich) in alpha Minimum Essential Medium (α-MEM; Biochrom, Berlin, Germany) for 2 h with shaking at 37°C and under 5% CO₂ in an incubator, the bones were placed in six-well plates in α-MEM supplemented with 10% fetal bovine serum superior (Biochrom), 1% penicillin/streptomycin, 1% L-glutamine and 0.5% amphotericin B (Thermo Fisher Scientific) in a 37°C and 5% CO₂ incubator. Mesenchymal progenitor cells (MSCs) were isolated from the long bones of mice as described previously (Rapp et al., 2018 (link)). Bone marrow cells were seeded at a density of 5.5×10⁷ cells/cm² in expansion medium (MesenCult™ Expansion Kit, Mouse, Stemcell Technologies, Vancouver, Canada). According to the manufacturer's instructions, the MSCs were cultured with additional MesenPure™ at 37°C and under an atmosphere of 6.0% O₂ and 8.5% CO₂. Osteoblasts and MSCs in passages 3-5 were used for further experiments.