Renilla glo luciferase kit

Pre-challenge serum was collected via submandibular bleed 2-3 days prior to RSV challenge and separated using Gel-Z Serum Separator Tubes (Sarstedt, Germany). Serum was stored at -80°C until heat inactivation (56°C for 30 minutes) and neutralizing antibody titers were performed. Serial dilutions of heat inactivated serum (50 mcL in phenol-free MEM supplemented with 5% FBS and Pen/Strep, Invitrogen) were incubated for 2 hours in a 37°C CO₂ incubator in a 96-well plate format with 100 pfu/well Line 19 RSV-Renilla Luciferase virus (provided by the Moore laboratory) in 50 mcL phenol free MEM medium as above. After 2 hours, Hep-2 cells were trypsinized and a total of 2.5 x 10⁴ cells were added per well in 25 mcL of phenol free MEM with FBS and antibiotics as above. Cells were incubated for a total of 64-66 hours at 37°C at 5% CO₂ and the luciferase readout was then obtained using the Renilla-glo luciferase kit (Promega) according to the manufacturer’s instructions. Luciferase activity (luminescence) was measured using a Novostar plate reader after a 15-minute incubation at 25°C. All plates were run in duplicate and averaged.

Pre-challenge serum was collected via submandibular bleed 2–3 days prior to RSV challenge and separated using Gel-Z Serum Separator Tubes (Sarstedt, Germany). Serum was stored at −80°C until heat inactivation (56°C for 30 min) and neutralizing antibody titers were performed. Serial dilutions of heat inactivated serum (50 mcL in phenol-free MEM supplemented with 5% FBS and Pen/Strep, Invitrogen) were incubated for 2 h in a 37°C CO₂ incubator in a 96-well plate format with 100 pfu/well Line 19 RSV-Renilla Luciferase virus (provided by Martin Moore) in 50 mcL phenol free MEM medium as above. After 2 h, Hep-2 cells were trypsinized and a total of 2.5 × 10⁴ cells were added per well in 25 mcL of phenol free MEM with FBS and antibiotics as above. Cells were incubated for a total of 64–66 h at 37°C, 5% CO₂ and luciferase readout was then obtained using the Renilla-glo luciferase kit (Promega) according to the manufacturer's instructions. Luciferase activity (luminescence) was measured using a Novostar plate reader after a 15 min incubation at 25°C. All plates were run in duplicate and averaged.