Ctl s6 analyzer

IFNγ ELIspot assay was performed by using a Murine IFNγ Single-color Enzymatic ELIspot Assay Kit (Cellular Technology Limited, Cleveland, OH, USA) in accordance with the manufacturer’s instructions. Freshly isolated lung cells (0.5–1.0 × 10⁵ per well) were incubated in CTL-Test Medium (Cellular Technology Limited) containing 2 mM glutamine (Medical & Biological Laboratories, Nagoya, Japan) and 10 µg ml⁻¹ peptide-25, an I-A^b-restricted CD4 epitope of Ag85B protein (aa240–254) (29 (link)) (Sequence; FQDAYNAAGGHNAVF; GenScript, Tokyo, Japan) overnight at 37°C in a 5% CO₂ atmosphere in an incubation chamber (Cosmo Bio). The number of spot-forming cells was determined by using a CTL S6 Analyzer, ImmunoSpot software (version 5.0) and Immuno Capture software (version 6.3) (Cellular Technology Limited).

Recombinant Cedar virus (rCedV) chimeras displaying RBP and F proteins of HeV or NiV_B were generated and validated as described elsewhere. Black-walled 96-well plates (Corning Life Sciences; NY, USA) were coated with 20,000 cells/well Vero E6 cells in DMEM + 10% Cosmic calf serum and incubated overnight. Approximately 24 hours later, HENV-103 and HENV-117 were diluted to indicated concentrations and incubated 1:1 with 4,000 PFU/well rCedV-HeV-GFP or rCedV-NiV_B-GFP and incubated for 2 hours at 37°C. Following incubation, 90 μL virus/antibody mixtures were added to aspirated cell monolayers and were incubated at 37°C for 22 hours. Medium containing virus/antibody mixtures was aspirated, and cells were fixed with 100 μL/well 4% formalin for 20 minutes at room temperature. After aspiration, cell monolayers were gently washed 4x with DI water. Formalin-fixed plates were then scanned using the CTL S6 analyzer (Cellular Technology Limited; OH, USA). Fluorescent foci were counted using the CTL Basic Count feature and S6 software. Percent neutralization was calculated by normalizing counts to a virus only control. Matrices were then imported into SynergyFinder and analyzed as described before.