Elisa plate reader

The MTS metabolic
catalytic assay is used to determine cell viability after exposure
to the hybrid nanomaterial. A549 and HDF cell lines are seeded in
different 96 well plates within the replicates at 5000 cells/plate
in a total volume of 150 μL, and the plates are incubated at
37 °C. Afterward, nanomaterials were injected rapidly to elucidate
the potential cytotoxicity of the probes for 24, 48, and 72 h. After
the treatment, the old medium is aspirated with a fresh solution of
4.5 g/L d-glucose in PBS mixed with the MTS reagent. After
1 h of incubation, the absorbance values of each well are determined
by using an ELISA plate reader (BioTek Instruments) at 490 nm.

The total flavonoid contents (TFCs) of kefir water samples were measured following a method described by Saeed et al. [36 (link)] with some adjustments. Nine microlitres of 5% NaNO₃ was mixed with 150 µL of kefir water samples (50 mg/mL) or catechin standard and incubated for 6 min in the dark. Then, 9 µL of 10% AlCl₃ was added and incubated for 5 min. After that, 60 µL of 1M NaOH and 72 µL of distilled water were added and mixed well by vortexing. The absorbance was then read at 430 nm using an ELISA plate reader (Bio-Tek Instruments, Winooski, VT, USA). The TFCs of the samples were expressed as µg catechin equivalents (µg CAT/µL). This experiment was performed in triplicates.

ELISA was performed to quantify the vaccine-specific IgG titers in mice sera. Briefly, ELISA plates were coated with recombinant trimeric HA derived from the A/Michigan/45/2015 H1N1 virus, which is closely related to IVR-180, as previously described (17 (link)), equivalent to 2 µg H1N1-HA/mL, in bicarbonate buffer and left overnight at 4°C. Plates were washed three times with 1× PBS and incubated in 100 μl of blocking buffer per well [5% fat-free milk in PBST (1× PBS + 0.1% Tween20)] for 1 h at room temperature (RT). In the meantime, the serum samples were serially diluted 3-fold, starting with 1:100 dilution, in blocking buffer and 50 μL of each sample dilution was incubated on HA-coated ELISA plates overnight at 4°C. The following day, the plates were washed three times with PBST and incubated with 100 μL of diluted horseradish peroxidase (HRP)-conjugated anti-mouse secondary total IgG (1:5,000) or IgG1 (1:2,000) or IgG2a (1:2,000) antibodies, for 1 h at RT. Finally, the plates were washed three times in PBST and incubated with 100 µl of tetramethylbenzidine (TMB) substrate at RT until the blue color appeared. The reaction was terminated with 50 µl of 1 M sulfuric acid (H₂SO₄), and the absorbance was measured at 450 nm and 650 nm wavelengths using BIOTEK ELISA plate reader.

A CCK8 kit (Dojindo, Shanghai, China) was used to perform the CCK8 assay following the manufacturer's protocol. Briefly, cells were resuspended and inoculated into a 96-well plate at a density of 1 × 10⁴ cells per well. The CCK8 reagent was added to the plate, followed by incubation at 37°C for 2 h. Finally, the absorbance was measured at OD 450 nm using an ELISA plate reader (BioTek, Winooski, VT, USA).