Multiskan mk3 elisa reader

Mice were randomly divided into two groups: the OVA-sensitized group (OVA) and the OVA-sensitized and PCs-treated group (OVA + PC). Mice in each group were administered 20 μg OVA (Sigma-Aldrich) by subcutaneous injection on days 0 and 1448 (link). Additionally, the OVA and OVA + PC groups were treated with saline or 1 × 10⁶ peritoneal cells, respectively. Whole blood was collected from the retro-orbital venous plexus on days 10 and 21. The amount of OVA-specific IgG in serum was determined by ELISA. Briefly, 96-well plates were coated with OVA at 10 μg/well overnight. After the plates were blocked with 5% milk (Bio-Rad), 100 μl/well of diluted serum was added and the plates were incubated at 37 °C for 2 hours. After incubation, plates were washed five times with PBST (PBS containing with 0.5% Tween-20 buffer) and the secondary antibody (goat anti-mouse IgG, diluted at 1:10000, Abcam, MA, USA) was added. Plates were incubated for 1 hour at 37 °C. After plates were again washed, 100 μl TMB (KPL, USA) was added and samples were incubated in the dark at room temperature for 20 min. The reaction was stopped by adding 100 μl/well of 2N H₂SO₄ and measured at 450 nm using a microplate autoreader (Multiskan Mk3 ELISA reader, Thermo).

An MTT assay was applied to measure the in vitro cytotoxicity of the {(Au⁰)₁₀₀G5.NH₂-FI-DOTA(Mn)-HA} NPs in a routine culture environment with 10% FBS (heat-inactivated, 1% penicillin-streptomycin) and 5% CO₂ at 37 °C.
HCCLM3 cells suspended in medium were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well with 200 μL per well and were incubated overnight. NPs with an Mn concentration ranging from 0–100 μg/mL were added into each well, and the cells were incubated for an additional 24 h. The mixture was then carefully removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 20 μL of MTT solution (5 mg/mL in PBS) was added into each well, and the cells were cultured for another 4 h at 37 °C and 5% CO₂. To dissolve the insoluble formazan crystals, the medium was carefully discarded and replaced with 200 μL of DMSO. Finally, the absorbance of each sample was measured at 570 nm using a Thermo Fisher Scientific Multiskan MK3 ELISA reader (Thermo Fisher Scientific, Hudson, NH).
The morphology of the HCCLM3 cells was observed to evaluate the cytotoxicity of the {(Au⁰)₁₀₀G5.NH₂-FI-DOTA(Mn)-HA} NPs after the cells were treated with the NPs at Mn concentrations of 0, 10, 20, 50, 75, and 100 μg/mL for 24 h. A Leica DM IL LED inverted phase-contrast microscope was employed to observe the cell morphology of each sample at a magnification of 200×.

The cell lines (brain endothelial bEnd.3 cells and rat C6 glioma cells) presents in this study were obtained from American Type Culture Collection (ATCC, United States). The bEnd.3 and C6 cancer cells were incubated with Fe₃O₄-Ce6 nanoparticles at different concentrations for 24 h, and then the cells were washed with PBS. The cells were then incubated in cell culture medium containing CCK-8 agent for 2 h. The supernatant of treated cells was collected to measure the absorbance at 450 nm using a Thermo Scientific Multiskan MK3 ELISA reader (Thermo scientific, United States), and then the cell viabilities were calculated.