Mycostrip

Stably transfected Chinese hamster lung (CHL) cells expressing the glucagon-like peptide-1 receptor (CHL GLP-1R, a kind gift of Prof. Brigitte Lankat-Buttgereit, Marburg, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/L D-glucose. Fetal calf serum (FCS, 10%), 2 mM L-glutamine, 0.5 mg/mL geneticin sulfate, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids and antibiotics (100 IU/mL penicillin, 0.1 mg/mL streptomycin, 0.25 μg/mL fungizone) were added and the cells were maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO₂. Hamster origin of the CHL cell line was confirmed and the cell line was tested negative for mycoplasma (MycoStrip™, InvivoGen).

MRC5 primary human fibroblasts (ATCC CCL-171, passage numbers 18–28) were used for all experiments. Cells were grown in complete growth medium (DMEM supplemented with 10% fetal bovine serum) under standard conditions (37°C and 5% CO₂). Cells were tested for mycoplasma using the MycoStrip (Invivogen) Mycoplasma Detection Kit and were authenticated by cell morphology and growth curve analyses.
Human cytomegalovirus infections were performed using AD169 or TB40/E virus strains. P0 virus was produced by transfecting fibroblasts with bacterial artificial chromosomes containing the viral genome, as described in references 124 (link), 125 (link). To generate P1 virus, which was used for all experiments, fibroblasts were infected with P0 virus, and the P1 virus produced was collected and concentrated by ultracentrifugation. Virus titer was determined by tissue culture infectious dose (TCID50).

HeLa cells were obtained from American Type Culture Collection and maintained in 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Gibco) and 50 μg/mL gentamicin (Nacalai Tesque). HeLa cells tested negative for mycoplasma using the MycoStrip (Invivogen, Cat #: 20596-84).

The mouse myogenic C2C12 cell line was obtained from ATCC (http://www.atcc.org/) and cells were used until passage number 20. Myoblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator kept at 37 °C and 5% CO₂. C2C12 myotubes were obtained by culturing 70% confluent myoblasts in differentiation medium (DMEM, 2% horse serum, 1% penicillin-streptomycin) for at least 4days. Authentication of the cells can rely on that done by the supplier, as most were used within a year of their purchase. We also authenticated C2C12 cell lines based on the morphology observed during experiments. Indeed, these cells turned to multinucleated myotubes upon differentiation, the formation of which is a unique feature of C2C12 cell lines. The cells were tested for Mycoplasma contamination with a detection kit (MycoStrip, Invivogen) that uses isothermal polymerase chain reaction (PCR) and can detect over 95% of commonly occurring mycoplasma species contaminating cell line. Results were negative.

The following cell lines were purchased from the ATCC: HEK293T/17 (CRL-11268), a transfectable cell line for virus production, and CRFK (CCL-94), a cat fibroblast cell line. Cell supernatants were tested for mycoplasma contamination by MycoProbe Kit (R&D Systems) or MycoStrip (InvivoGen). Cells were grown on tissue-culture treated plates in DMEM containing high glucose and L-glutamine (Gibco) and supplemented with 1x penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were grown at 37°C in 5% CO₂ in humidified incubators. Cells were harvested from plates by digestion with 0.05% trypsin-EDTA (Thermo Fisher) and counted using a TC20 (Biorad) or Vi-Cell BLU (Beckman) automated cell counter.

THP1-Lucia ISG and IFN-α/β Reporter HEK 293 Cells were purchased (Invivogen, San Diego, CA, USA); the NS-SV-TTAC Acinar Cells (aka: AC cells) were provided by Dr. Jay Chiorini. THP1 and 293 cells were cultured as previously reported in RPMI (Corning Cellgro, Corning, NY, USA, #15–040-CV) and DMEM (Gibco, New York, USA, #10313–021), respectively, supplemented with 10% FBS (Gibco, #A47668–01) and penicillin/streptomycin 100 U/mL, 2 mM L-glutamine, 100 ug/mL Normocin. The NS-SV-TTAC cell line was cultured in Defined K-SFM (ThermoFisher, #10744019), supplemented with Defined Keratinocyte-SFM Growth Supplement. All cell lines were maintained in a humidified 37° C incubator with 5% C0₂, and cell lines were tested and confirmed negative for mycoplasma, validated with MycoStrip (InvivoGen, San Diego, CA, USA, #rep-mys-50).