Phosphate buffered saline (pbs)

Mandibular incisors from SD rats were immersed in phosphate-buffered solution (PBS) (Biosharp, Beijing, China) containing 1% double-antibody. Pulp tissues were obtained and digested in trypsin + 3 mg/ml type I collagenase for 50 min. The resulting mixture was centrifuged and added to a specialized medium for stem cells in T25 flasks. The flasks were incubated at 37 °C with 5% CO₂ for 2–4 generations before being used for subsequent experiments.

The apoptosis rate was detected using an Annexin V-FITC/PI Apoptosis Detection Kit (BD, USA). The cells were collected and washed twice with PBS (Biosharp, Beijing, China), which was followed by gentle suspension in 500 μL of 1× Annexin V Buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solution was added to the cells, which were then incubated at 24 °C for 15 min while protected from light. The cell cycle distribution of granulosa cells (GCs) was determined using a cell cycle assay kit (KeyGEN, Nanjing, China). After collection and two washes with PBS, 500 μL of PI/RNase staining buffer was added to each tube of cell samples. The cells were gently resuspended and incubated for 15 min at 37 °C in the absence of light. Finally, the apoptosis rate and cell cycle distribution were analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA) and Flowjo software (version 7.6).

Terminal‐deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining in cultured spinal cord neurons were performed. Briefly, the samples were collected on the 3rd days after transfection, cultured neurons were fixed with 4% paraformaldehyde (Cat# 142287, Beyotime, Shanghai, China) for 20 min at room temperature, then rinsed with PBS (Cat# BL551A, Biosharp, Hefei, China) for 20 min. Subsequently, cells incubated with TUNEL reaction mixture (Cat# Rs‐11684817910, In situ Cell Death Detection Kit, Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at 37°C, then rinsed with PBS for 3 times and finally incubated with DAPI (Cat# C1005, Beyotime Biotechnology, Shanghai, China) for 5 min. Five fields were acquired, and apoptosis was quantified by determining the percentage of TUNEL/DAPI with Leica AF6000 DMI6000B (LAS AF system).

The protein adsorption of Ti, TNTs, and TNT-GO was detected using bovine serum albumin (BSA, Sigma-Aldrich Co., St. Louis, MO, USA) as a model protein. Each group of samples was placed in a 24-well plate, and 200 μL of protein solution (2 mg/mL BSA) was added. After incubation for 1 h, 20 μL of supernatant was collected from each well to evaluate the amount of unbound protein. The samples were washed thrice with phosphate-buffered saline (PBS, Biosharp, Hefei, China). Then, 200 μL of 2% sodium dodecyl sulfate (SDS, Beyotime, Shanghai, China) was added to the sample surface to elute proteins under shaking for 2 h. A BCA protein detection kit was used to detect the protein concentration. A microplate spectrophotometer was used to determine the optical density (OD) at 562 nm.

The paired serum samples were tested in parallel by HAI assays using 1% red blood cells from turkey or guinea pigs to measure the endpoint of HAI antibody titers against all vaccine strains. According to the standard reagent preparation protocols from WHO, influenza virus antigens were cultured by specified pathogen-free (SPF) chicken embryo. Serum antibody titers were ascertained as the highest fold of dilution which could completely lead to HAI, and 60 µl receptor-destroying enzyme (RDE, Denka Seiken, Japan) was added to 20 µl serum to remove the non-specific inhibitors in 37°C water-bath for 16–18 h. Then, the compound was inactivated by 56°C water bath for 30 min before adding 20 µl phosphate buffered saline (PBS, Biosharp, China). The serums were tested in two-fold serial dilutions with an initial fold of 1:5.
The seroconversion rate was defined as the percentage of participants mounting to at least 1:40 in the postvaccination titer from the pre-vaccination titer less than 1:10 or achieving at least four-fold increase in the postvaccination titer to the pre-vaccination antibody titer. Responder was defined as those who achieved seroconversion to all vaccine composition strains, and non-responder was those who did not achieve seroconversion to all vaccine composition strains. According to the definitions, 365 responders and 227 non-responders were selected for genotyping.

Briefly, after being washed twice with PBS (Biosharp) and detached with 1 mL of 0.25% trypsin (Mengbio), adherent cells at a density of 1 × 10⁶ were harvested. Then, BMMSCs were resuspended. The cells were incubated with rat CD29 (PE), CD90 (FITC), CD31 (PE) and CD45 (PE) (BD Bioscience) antibodies at 4°C in the dark. Finally, the samples were assessed using a flow cytometer.

HEK 293 T cells grown in 12-well plates (Corning) were cotransfected with pHA-NCL and p3 ×Flag-N. After 36 h transfection, cells were fixed with 4 % paraformaldehyde in PBS (Biosharp) for 30 min and permeabilized by 0.1 % Triton X-100 for 15 min. The cells were blocked with 5 % nonfat milk in PBS overnight at 4 °C and subsequently incubated with primary antibodies (mouse anti-HA MAb, 1 : 1000; rabbit anti-Flag MAb, 1 : 1000) for 1.5 h at 37 °C. After being washed five times for 5 min each time, the cells were incubated with secondary antibodies [Alexa Fluor 488 goat anti-rabbit IgG (H+L), 1 : 2000; Alexa Fluor 594 goat anti-mouse IgG (H+L), 1 : 2000] for 1 h. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) for 15 min and observed using the Zeiss SP2 confocal system (Leica Microsystems, Germany).