Hek blue detection reagent

HEKBlue cells were seeded into 96-well plates (50.000 cells/well). After 24 h, the respective cell culture media was replaced by HEK-Blue Detection reagent (InvivoGen #hb-det2, San Diego, CA, USA), which was prepared according to the manufacturer’s instructions. Next, cells were incubated with 10 µg/ml of SARS-CoV-2 RNA-fragments or control oligonucleotide complexed to LyoVec for 24 h. The NF-κB/AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter protein was detected using the SpectraMax iD3 (Molecular Devices, San Jose, CA, USA) at a wavelength of OD 655 nm.

HEK293 cells expressing human/mouse TLR7/8 NF-κB/Secreted Embryonic Alkaline Phosphatase (SEAP) reporter and their respective parental control cell lines were used for TLR activation assays. All cell lines were purchased from Invivogen. A total of 30,000 cells were seeded onto 96-well plates in DMEM. The following day, the media was changed to 90% HEK-Blue detection reagent (InvivoGen #hb-det2) and 10% DMEM. The cells were then incubated with indicated miRNAs (10 μg/mL) complexed with Lyovec, or controls for 24 h. All conditions were performed in triplicate and analysed at a wavelength of 655 nM using a SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).

Human TLR7 and human TLR8/NF-κB/Secreted Embryonic Alkaline Phosphatase (SEAP) reporter HEK293 cells were used for TLR activation assays. The parental control cell lines HEK-Blue Null-1 k and Null1, respectively, were used as control (all lines obtained from InvivoGen, San Diego, CA, USA). Cells were seeded into 96-well plates (5 × 10⁴/well). After 24 h, cells were incubated with the synthetic oligoribonucleotides or control oligonucleotide complexed to the transfection agent LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA) according to the manufacturer’s instructions. Cells were stimulated with indicated agents dissolved in 90% HEK-Blue detection reagent (InvivoGen #hb-det2, San Diego, CA, USA) and 10% cell culture media. Each condition was performed in triplicate. The reporter protein SEAP was detected using the Varioskan Flash device (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of OD 655 nm.