96 well polystyrene plate

Influenza virus antigen-specific antibodies were measured by ELISA. Briefly, polystyrene 96-well plates (Nunc, China) were coated with whole ITIV antigens (HA, 1 μg/mL per strain) in 0.1 M bicarbonate buffer (pH 9.5) and incubated overnight at 4°C. The plates were washed with PBS-0.05% Tween-20 (PBST) and then blocked with PBS-1% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 2 h at 37°C. After washing with PBST, 100 μL of the samples serially diluted with PBS-0.1%BSA was added, and the plates were incubated for 2 h at 37°C. After washing with PBST, an HRP-conjugated goat anti-mouse IgG or IgA antibody (Sigma-Aldrich, USA) was added at a 1:5000 dilution and incubated overnight at 4°C. A color reaction was developed using tetra-methyl benzidine substrate solution (Sigma-Aldrich, USA) at 37°C for 1 h. The reaction was stopped by adding 0.05 mL of 0.5 M H₂SO₄ per well, and the optical densities (ODs) were read at 450 nm using a microplate reader (Thermo Scientific Multiskan GO, USA). The antibody titers were expressed as the reciprocal of the highest dilution of sample for which the OD was 5×OD_{mean background}, and the geometric mean titer (GMT) was calculated.

ELISA was performed using the DuoSet Kits from the R&D, as previously described [26] (link), [44] (link). Briefly, polystyrene 96-well plates (Nunc) were pre-coated overnight at RT with specific capture antibody, then blocked with 1% BSA in PBS for 1 h at RT, and incubated with standard cytokine dilutions and cell cultures media (100 µl, undiluted) for 2 h at RT followed by washes with PBS plus 0.1% Tween 20, and incubation with biotinylated detection antibody for 2 h at RT. After the second wash the plates were incubated with HRP-streptavidin for 20 min at RT and washed again. The signal was developed after addition of 3,3′,5,5′-tetramethylbenzidine-peroxidase EIA kit (Bio-Rad) until color appeared and the reaction was stopped by 1 M H₂SO₄. Microplate reader (Dynex Technologies) was used to detect the signals with 450 nm and correction at 530 nm. The detection range of IL-1β ELISA was 3.9–250 pg/ml.

The cytotoxicity of the NO/THCPSi NPs was evaluated using NIH/3T3 fibroblast
cells. The cells were maintained in DMEM supplemented with 10% FBS and
2 mM l-glutamine, 100 U/mL penicillin,
100 μg/mL streptomycin, and incubated at 37°C with 5%
CO₂.
All mentioned procedures for the preparation of NO/THCPSi NPs and glucose/THCPSi
NPs were done under sterile conditions within a biological safety cabinet
(Bio-cabinet, Aura 2000, Microprocessor Automatic Control, Firenze, Italy).
The NIH/3T3 cells were trypsinized and then seeded into polystyrene 96-well
plates (Nalge Nunc International, Penfield, NY, USA) at a density of
3 × 10⁴ cells/mL and then after 24 h, the
cultured cells were incubated with NO/THCPSi NPs, glucose/THCPSi NPs, and THCPSi
NPs at four different concentrations from 0.05 to 0.2 mg/mL for
48 h.
After the incubation period, the culture medium was separated from the cultured
cells and subjected to a LDH assay that was carried out following the
manufacturer’s instructions. Moreover, a FDA-PI assay was performed on the
cultured cells remaining in the wells. The cells were incubated with fresh
medium before adding final concentrations of 15 μg/mL FDA and
5 μM PI for 3 min at 37°C to count the live and dead cells,
respectively, using a fluorescence microscope (Eclipse, Ti-S, Nikon, Tokyo,
Japan) and determine the percentage of live cells. All experiments were repeated
at least three times.