Gentamycin

The cell line HepG2 was used in both in the MTT test and the in vitro mammalian chromosome aberration test. Cells were derived from human liver tumors, originally established by Dr. B.B. Knowles (Wistar Institute of Anatomy and Biology, Philadelphia, PA, USA) and kindly provided by A. Collins (Department of Nutrition, University of Oslo, Oslo, Norway). The cell line HepG2 is capable of activating many indirect mutagens by its expression of the cytochrome P-450 superfamily members (CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP2C19, CYP2A6, CYP2B6, CYP2C8, CYP2D6, and CYP2E1) [37 (link)]. This cell line is characterized by an aneuploid number of chromosomes. The HepG2 cell line was used at passage number 21. Cells were cultured in Wiliams medium (PAN-Biotech GmbH) with 10% fetal bovine serum (PAN-Biotech GmbH). The Williams medium was supplemented with antibiotic gentamycin (50 μg/mL) (PAN-Biotech GmbH). Cells were cultured in plastic Petri dishes (Ø = 60 mm) under CO2/air (5%:95%) at 37 °C, as described by Miadokova et al. [51 (link)].

The Vero cells (African green monkey kidney epithelial cells) and the BHK 21 cells (Baby hamster Kidney epithelial cells) were maintained in the Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech, Aidenbach, Germany) supplemented with 5% Fetal Bovine Serum (FBS, PAN Biotech, Aidenbach, Germany), 0.1% Gentamycin and 1% Penicillin–Streptomycin (PAN Biotech, Aidenbach, Germany). The Raw264.7 cells (mouse monocyte/macrophage cell line), was maintained in RPMI-1640 (Himedia Laboratories Pvt. Ltd, Mumbai, India) supplemented with 2.0 mM L-glutamine, Penicillin 100 U/mL, Streptomycin 0.1 mg/mL and 10% Fetal bovine serum (FBS; PAN Biotech, Germany) at 37 °C under a humidified incubator with 5% CO₂. The HSV-1 virus strain KOS with GenBank accession Number JQ673480.1 [15 (link)] was kindly gifted by Dr. Roger Everett, Glasgow University, Scotland. MBZM-N-IBT was synthesized by our group [13 (link)] and acyclovir was procured from Sigma (Sigma, USA). The anti-ICP8 (ab20194), anti-gC (ab6509) monoclonal antibodies and GAPDH antibody were procured from Abcam (Cambridge, UK) and Abgenex Pvt. Ltd. (Bhubaneswar, India) respectively.

Human embryonic kidney (HEK) 293T cells (RRID:CVCL_0063) (laboratory of Tobias Moser), SLK cells (RRID:CVCL_9569) (NIH AIDS Research and Reference Reagent program), rhesus monkey fibroblasts (RF) (laboratory of Prof. Rüdiger Behr) and HaCaT human keratinocytes (RRID:CVCL_0038) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, 25mM HEPES (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific), and 50μg/ml gentamycin (PAN Biotech). iSLK cells (laboratory of Don Ganem, Novartis Institutes for BioMedical Research, Emeryville, CA, USA) were maintained in DMEM supplemented with 10% FCS, 50μg/ml gentamycin, 2.5μg/ml puromycin (InvivoGen) and 250μg/ml G418 (Carl Roth). Raji cells (RRID:CVCL_0511) (laboratory of Jens Gruber), MFB5487 (a clonal cell line established from Macaca fascicularis PBMC, immortalized by infection with herpesvirus papio; a kind gift from Ulrike Sauermann) and MMB1845 cells (a clonal cell line established from Macaca mulatta PBMC, immortalized by infection with herpesvirus papio; a kind gift from Ulrike Sauermann) were cultured in RPMI (Thermo Fisher Scientific) supplemented with 10% FCS and 50μg/ml gentamycin.

Adult A. suum worms were collected bimonthly from a slaughterhouse in Brandenburg (Germany) post-slaughter. Collections were made on days when primarily organic animals were slaughtered. Each batch of worms contained 100–300 worms of different sizes from different pigs. Motile, female worms were separated and washed several times in pre-warmed (37 °C) 0.9% NaCl solution. Worms were transferred into glass bottles at a density of four worms per 200 ml of Hanks' Balanced Salt Solution supplemented with antibiotics (HBSS-AB: 127 mM NaCl, 7.5 mM NaHCO₃, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ × 6 H₂O), supplemented with 200 U/ml penicillin, 200 μg/ml streptomycin, 2.5 μg/ml amphotericin B, and 50 μg/ml gentamycin (PAN-Biotech GmbH, Germany), and cultured at 37 °C and 5% CO₂. After 24 h, worms were transferred into fresh culture medium, and excreted eggs were harvested and collected on a 30-µm cell strainer (Miltenyi Biotec) and washed by flushing the strainer with 50 ml HBSS. Subsequently, eggs were embryonated for 6–8 weeks at 33 °C in 50 ml distilled water (dH₂O) containing 0.1% formaldehyde and protected from light. The embryonation status was assessed microscopically.

In 96-well cell plates, 5 × 10⁴ CD4⁺ T cells/well were cultured in 200 µl DMEM + GlutaMAX™ (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific), 50 µg/ml gentamycin (PAN Biotech, Aidenbach, Germany), 10% (v/v) heat-inactivated fetal bovine serum (Biowest, Riverside, MO, USA) and 50 μM 2-Mercaptoethanol (PAN Biotech) in the presence of 5 × 10⁴ CD3-Biotin/CD28-Biotin/CD2-Biotin labeled anti-Biotin MACSiBead^TM particles and 400 U/ml IL-2 (Proleukin®, Novartis Pharma, Nuremberg, Germany) for 120 h, following a 24 h resting phase by replacement of the cell culture media with 200 µl new DMEM without any cytokines. Notch activation via DLL4 was induced by addition of 15 × 10⁴ MACSi Beads/well, covalently coated with recombinant mouse DLL4 (rmDLL4-hFc, kindly provided by Antonius Rohlink, Basel, Switzerland) from transfected CHO cells. If indicated, cytokines IFN-α, IL-6, IL-21 were added to the cell culture medium at 20 ng/ml. If not mentioned otherwise, all reagents were purchased from Miltenyi Biotec.

Vero cells, originated from African green monkey kidney epithelial cells; S 27, Chikungunya virus prototype strain (accession no. AF369024.2) DRDE06, 2006 Indian ourbreak strain (accession no. EF210157.2) and E2 monoclonal antibody for CHIKV were gifted by Dr. M. M. Parida, DRDE Gwalior, India. Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech, Aidenbach, Germany) was used to maintain the Vero cell line supplemented with 5% Fetal Bovine Serum (FBS, PAN Biotech, Aidenbach, Germany), Gentamycin and Penicillin-Streptomycin (PAN Biotech, Aidenbach, Germany). The anti-CHIKV-nsP2 monoclonal antibody was developed by us52 (link) and GAPDH antibody was procured from Imgenex India (Imgenex, Bhubaneswar, India). MIBT was synthesized by us during this study and Ribavirin was procured from Sigma (Sigma, USA).

The JA cell cultures were generated from at least 1 cm³ of native tumor samples, as described previously [30 (link)]. Briefly, the tissue samples were mechanically dissected and cultivated in DMEM F/12 Glutamax (Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Darmstadt, Germany), 1% penicillin–streptomycin, 1% sodium pyruvate, 10 µg/mL gentamycin (PAN-Biotech GmbH, Aidenbach, Germany) and 2.5 µg/mL amphotericin B (Thermo Fisher Scientific, Darmstadt, Germany) at 37 °C and 5% CO₂. The medium was changed twice a week and the cells were passaged at 70% confluence by a split ratio of 1:3 with accutase solution (PromoCell, Heidelberg, Germany).