Chicken red blood cells rbcs

H1N1 IAV at 10 MOI and IQC (0, 1, 5, 10, 25 µM) were mixed for 1 h at 4 °C and the mixtures were added to RAW 264.7 cells in 12 wells (5 × 10⁵ cells/well) for 2 h at 37 °C. After washing with PBS, the cells were further incubated for 24 h at 37 °C. The supernatant was harvested for the hemagglutination assay. Briefly, each supernatant was serially two-fold diluted and added to a round-bottomed 96-well plate. The blank medium was used as a negative control. Each sample (100 µL) was mixed with an equal volume of 1% chicken red blood cells (RBCs) (Innovative Research, Inc., Southfield, MI, USA) in PBS, and incubated for 1 h at room temperature. RBCs in negative control exhibited agglutination and RBCs in the virus-infected well were lysed without agglutination. HA titers were calculated as HA unit/100 µL in comparison with values of the virus control treatment. HA units were determined as the point at which hemolysis occurs.

Template (containing 30 random bases), primers, and all oligonucleotides were synthesized by Bioneer (Daejeon, Korea). Pfu DNA polymerase was purchased from Biofact (Daejeon, Korea), and restriction enzymes and DNA ligases were purchased from Takara Bio (Shiga, Japan). Chicken red-blood cells (RBCs), packed at 5%, were purchased from Innovative Research (Detroit, MI, USA). Allantoic fluid and influenza A virus subtypes (H5N1 (A/wild duck/Korea/SNU50-5/2011), H1N1 (A/Puerto Rico/8/1934), H9N2 (A/Duck/Hong Kong/702/1979), recombinatnt H5N1(p), and recombinant H5N6) were provided by the Seoul National University, College of Veterinary Medicine. M13 phage was purchased from New England Biolabs (Ipswich, MA, USA). Reduced graphene oxide (rGO) was purchased from Graphene Supermarket (Ipswich, MA, USA).