Pen strep

Human bone marrow-derived mesenchymal stromal cells (MSCs) and cardiomyocyte progenitor cells (CPCs) were obtained and isolated as described before [34 (link),35 (link)]. MSCs were cultured in MEM-alpha (Gibco, 32561–037) supplemented with 10% fetal bovine serum (Gibco, 10270–106) + 1% PenStrep (Lonza, 17-602E) + 0.2 mM L-ascorbic acid-2-phospate (Sigma A4034) + 1 ng/ml bFGF (Sigma F0291). CPCs were cultured in SP++ (25% EGM-2 (Lonza CC-3156) + 75% M199 (Gibco 31150–022) supplemented with 10% fetal bovine serum + 1% PenStrep + 1% non-essential amino acids (Lonza 13–114). Cultures were incubated at 37°C (5% CO₂ and 20% O₂) and adherent cells were passaged when reaching 80–90% of confluency using trypsin digestion (0.25%, Lonza, CC-5012). MSCs and CPCs from fetal or adult donors were used in the co-cultures between passage 6–17.

FreeStyle^TM 293F (HEK-293F), Expi293F^TM cells (Expi) and FreeStyle^TM CHO-S (CHO-S) cells were cultured in FreeStyle^TM 293 expression medium and FreeStyle^TM CHO expression medium, respectively (Invitrogen). Additional cell lines were obtained from the American Type Culture Collection (ATCC). B16-F10 (C57Bl/6-derived mouse skin melanoma) cells were cultured in DMEM medium containing Ultraglutamin 1 (Lonza), supplemented with 10% (v/v) Donor Bovine Serum with Iron (Life Technologies) and Pen/Strep (Lonza). Raji (human CD20-positive Burkitt’s lymphoma) cells and Daudi (human CD20-positive Burkitt’s lymphoma) cells were cultured in RPMI 1640 medium (Lonza), supplemented with 10% (v/v) Donor Bovine Serum with Iron, 1% (w/v) L-glutamine 200 mM in 0.85% (w/v) NaCl solution (Lonza), 1% Sodium Pyruvate (Lonza) and Pen/Strep (Lonza). All cell lines were maintained at 37 °C in a 5% CO₂ humidified incubator.

Cells were maintained in a 37 °C incubator with 5% CO2. HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% foetal bovine serum (FBS) (Gibco) and 5 U ml^− 1 Penstrep (Lonza). Neuro-2a(N2A) (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% FBS (Gibco), 5 U ml^− 1 Penstrep (Lonza) and 5 mM sodium pyruvate.
HEK293T and N2A cells were transfected with 700 ng of plasmid using 3.5 μg PEI/ml media and one tenth media volume of OptiMEM in a 24 well format. Approximately, 50,000 HEK293T cells were seeded / well and 75,000 N2A cells were seeded per well of the 24 well plate. Proteins were extracted 72 h post-transfection. Cells were washed in ice cold phosphate buffered saline (PBS) and subsequently lysed in ice cold lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM EDTA, 1 mM DTT, protease inhibitor cocktail (Sigma)) for 10 min on ice. Extracts were then centrifuged at 17,000 g for 5 min at 4 °C. Extracts were quantified using Bradford Reagent (BioRAD), resolved by SDS-PAGE, electroblotted onto nitrocellulose membrane and probed to the relevant primary antibodies.

Hela R19, HEK293T and Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) supplemented with 10% fetal bovine serum (FBS) and 1% Pen-Strep (Lonza). BHK21 cells were cultured in DMEM containing sodium pyruvate and glutamax (Gibco; 2206106) and supplemented with 10% FBS and 1% Pen-Strep (Lonza).
CVB3 was generated by passaging the virus on Hela R19 cells. Upon complete CPE, the virus was harvested and concentrated by ultracentrifugation (30% sucrose, 140.000 g for 16 hours, 4°C, SW32Ti rotor). The virus was subsequently diluted in PBS and stored at -80C. EMCV and Renilla luciferase (Rluc)-CVB3 viruses were generated by producing RNA from their respective infectious clones and transfecting this RNA into BHK21 or HEK293T cells respectively. Similar to the production of CVB3, viruses were harvested after complete CPE, concentrated by ultracentrifugation, diluted in PBS and stored at -80°C. Virus titers were determined by end point titration on Hela R19 and BHK21 cells according to the method of Spearman-Kärber and expressed as 50% Tissue Culture Infectious dose (TCID50).

Well described iPSC-derived neuronal progenitor cells, long term neuroepithelial-like stem cells (ltNES), lineages AF22 (WT) and ADP2 (V717I London APP mutant) were kindly provided by Anna Falk (Karolinska Institute, Sweden)^{14 (link)–21 (link)}. iPSC-derived cells were expanded in DMEM/F12 with Glutamax (Gibco), supplemented with EGF (10 ng/mL; Peprotech) and FGF2 (10 ng/mL; Peprotech), 1% N2 (Gibco), 0.1% B27 (Gibco), 1% Pen/Strep (Lonza). Once confluent, cells were differentiated for 60 days to obtain neuronal phenotypes^{14 (link)}, followed by the addition of 1 µM amyloid beta oligomers (oAβ) for 24 h. Next cells were thoroughly washed, trypsinized and lysed in 4% SDS lysis buffer supplemented with protease inhibitor (HALT protease inhibitor cocktail; Thermo Scientific) and phosphatase inhibitor (PhosSTOP; Roche). Cell lysates were stored at −80 °C until use. Cell differentiation media was as follows: 1:1 mixture of Neurobasal and DMEM/F12 with Glutamax, supplemented with 1.5% N2, 1% B27 (all from Gibco) and 1% Pen/Strep (Lonza), with ½ media changed every day. These cells have been demonstrated to maintain stable morphology, differentiation potential and gene expression profiles over more than 100 passages^{14 (link)}, however the cells used in this study did not exceed passage 40.

To identify the most dispensed medicines in Central Norway in 2019, we searched the Norwegian Prescription Database (www.norpd.no; accessed on 3 May 2019), which contains drugs, Anatomical Therapeutic Chemical (ATC) codes, and daily defined dosages (DDDs). As search criteria, we included all age groups and both sexes. Table S1 lists 45 active compounds of the most dispensed medicines, their suppliers, and their catalogue numbers. To obtain 10 mM stock solutions, compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Steinheim, Germany) or milli-Q water, depending on solubility. The solutions were stored at −80 °C until use. Human telomerase reverse transcriptase-immortalized retinal pigment epithelial (RPE, ATCC MBA–141) cells were grown in Dulbecco’s Modified Eagle’s F12 medium (DMEM-F12; Gibco, Paisley, Scotland) supplemented with 100 U/mL penicillin and 100 ug/mL streptomycin mixture (Pen/Strep; Lonza, Cologne, Germany), 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS; Lonza, Cologne, Germany), and 0.25% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA). Human influenza A/WSN/33(H1N1) virus was generated using the eight-plasmid reverse genetics system, as described previously [19 (link)].

JPCs derived from three donors were included in this study in accordance with the local ethical committee (approval number 074/2016BO2) and after obtaining written informed consent. Jaw periosteal tissue was extracted during routine surgery and JPCs were isolated and expanded as previously reported [13 (link)]. JPCs and iMSCs were grown in hPL5-medium (DMEM/F12 (Gibco) + 5 % human platelet lysate (hPL, ZKT Tübingen GmbH), 100 U/mL penicillin-streptomycin (Pen-Strep, Lonza, Basel, Switzerland), 2.5 µg/mL amphotericin B (Biochrom, Berlin, Germany)).
iPSCs were maintained in Essential 8 medium (E8, Thermo Fisher Scientific Inc., Waltham, MA, USA) with daily medium changes and passaged every 4–6 days using 10 µM Y27632 ROCK inhibitor (Selleck Chemicals LLC, Houston, TX, USA).