Apc anti mouse h 2kb bound to siinfekl antibody

OVA was used as a model antigen. Different concentrations of NPs (50 μl) were added to the OVA solution (50 μl, 0.5 mg/ml) and incubated at 37°C for 30 min. After that, the mixture was centrifuged (12,000g, 10 min), and the supernatant was collected. The concentration of supernatant was detected with the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). To evaluate the antigen cross-presentation ability of NPs, DC2.4 cells were seeded in 24-well plates (0.15 × 10⁶ cells per well) and cultured for 24 hours. Then, free OVA and OVA/NP complexes (OVA concentration of 5 μg/ml) were added and further cultured for 24 hours. Cells were collected and stained with the APC anti-mouse H-2K^b bound to SIINFEKL antibody (BioLegend) before washing and assessment by flow cytometry (BD FACSCanto II).

Cell viability of HeLa-K^b cells 20 h post infection was determined by Zombie Aqua staining (BioLegend, San Diego, CA, USA) followed by flow cytometry. The infection rate was calculated by flow cytometric detection of mCherry or Green Fluorescent Protein (GFP) expressing cells. Quantification of H-2K^b molecules on the surface of ORFV-infected HeLa-K^b cells was done using QIFIKIT (Dako, Glostrup, Denmark) according to the manufacturer’s protocol. Detection of Ova_257-264 SIINFEKL peptide on the surface of ORFV-infected cells was performed after staining with Allophycocyanin (APC) anti-mouse H-2K^b bound to SIINFEKL antibody (BioLegend, San Diego, CA, USA; clone 25-D1.16). Samples were acquired on a BD Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA).