ELISA plates were coated with 4 g/mL of A/Aichi/2/68 virus

ELISA plates were coated with 4 g/mL of A/Aichi/2/68 virus. previous described.16 Briefly, 36 mL of 1 1 mM chloroauric acid was kept well mixed under boiling conditions for 10 min. Then, 4 mL of 90 mM sodium citrate was quickly added to the solution. The solution was stirred for 20 min to allow full reduction. The reddish color solution was cooled down to room temperature. The solution was stored away from light at 4C. The size of AuNPs was confirmed by using a transmission electron microscope (TEM) and dynamic light scattering (DLS; Malvern Nano-ZS; Malvern Instruments, Malvern, UK). Ultraviolet (UV)Cvisible absorbance spectra were recorded at room temperature. Preparation of Alkyne-FliC Alkyne-FliC was prepared according to a modified EDC method.17 Briefly, 1 mL of 2.4 mg/mL FliC (PBS, pH =7.4), 12 L of 4-aminophenyl propargyl ether (100 mM in THF), and 11 L of EDC (100 mM in PBS) were mixed in 10% v/v THF/PBS overnight at 4C. The crude alkyne-FliC was purified by a Microcon centrifugal filter unit (molecular weight cutoff [MWCO] 10 kDa) and washed with PBS. Purified alkyne-FliC was stored under 4C. The molar concentration of alkyne-FliC was determined by Bradford protein assay.18 Preparation of dual-linker modified AuNPs Briefly, 1 mL (0.005 nmol) AuNPs solution was mixed with 10 L of 10 mM Tween 20 for 30 min to stabilize the particles. After adding 10 L of 1 1 mg/mL SH-NTA in DMSO, the solution was continually kept mixing for 2 h. Then, 100 L of 100 M azide-PEG-SH solution was added. Two more hours mixing was required to allow for the complete exchange of citrate with thiol. The mixture was purified by centrifugation (?32,869 for 30 min at 4C) twice. The supernatant was removed, ITGAV and the pellet was re-suspended in 1 mL 18.2 Mcm E-pure water. Preparation of AuNPs-HA/FliC conjugates via metal-chelating and click chemistry reactions Briefly, 33 L of 30 mM copper (II) sulfate pentahydrate and 17 L of alkyne-FliC (1.5 mg/mL) were added to 1 mL SH-NTA and azide-PEG-SH AuNPs and kept mixing for 30 min. After adding 140 L HA (1.8 mg/mL), the solution was mixed overnight to allow complete protein conjugation at 4C. The mixture was purified by centrifugation (30,563 for 20 min at 4C) twice. The pellet was redispersed in 1 mL PBS. The particles size was characterized by TEM and DLS. Quantification and stability of HA/FliC-conjugated Butabindide oxalate AuNPs Conjugated proteins were released from AuNPs by displacing the linker from particles using 0.1 mM 11-mercapto-1-undecanol and heating. Samples were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with Coomassie blue. Band densitometry, calibrated with an HA protein standard (Figure S1), was used to determine the mass of protein Butabindide oxalate released from AuNPs. The concentration was also confirmed by a Bradford protein assay. TLR5-specific bioactivity assay A HEK 293T (human embryonic kidney 293T, CRL-3216; American Type Culture Collection [ATCC], Manassas, VA, USA) cell-based assay with modifications was used to determine the bioactivity of conjugated FliC on AuNPs.19 HEK 293T cells were grown in Dulbeccos Modified Eagles Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; heat inactivated) (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific), and 1% penicillin/streptomycin. In brief, 90% confluent 293T cells in a 75 cm2 flask were transfected with 10 g of plasmid pUNO1-hTLR5 (Invivo-Gen, San Diego, CA, USA) and reporter plasmid pGL4.3 (Promega Corporation, Fitchburg, WI, USA) by using Lipofectamine? 2000 (Thermo Fisher Scientific) following the manufacturers instructions. The ratio between pTLR5 and pGL4.3 ranged from 5:1 to 10:1. After 24 h of transfection, cells were split into a 96-well plate with 5104 cells/well. Soluble FliC and AuNPs-HA/FliC were serially diluted from 2 g/mL to 1 1.6 ng/mL, were prepared with 1% FBS culture medium, and used to treat cells for stimulation. After 5 Butabindide oxalate h incubation, 100 L of Luciferase Assay Reagent (Promega Corporation) was added to each well. Luciferase activity was read with GloMax?-Multi Detection System (Promega Corporation). Hemagglutination assay Hemagglutination tests.