Elisa plate reader

Cytotoxicity of MNPs was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Briefly, 10⁴ HeLa/ MCF7 cells per well were seeded in triplicates in a 96-wells tissue culture plate and grown for 24 h followed by treatment with magnetic fluid diluted in EMEM in the concentration range varying from 0.75 to 0.03 mg/mL. Two sets of triplicates served as untreated controls.
After incubation the magnetic fluid was removed and the cells were washed thrice with phosphate buffered saline (PBS) to ensure complete removal of MNPs from the wells. Thereafter, 300 μl of media and 25 μl of MTT solution (5 mg/mL in PBS) was added to each well followed by 3 h of incubation. Subsequently, media containing MTT was removed and the formed crystal formazan were dissolved in 100 µl dimethyl sulfoxide. The MTT assay absorbance was measured at 570 nm on an ELISA plate reader (Molecular Devices, USA). After obtaining the IC₅₀ value we utilized the same assay to report our preliminary results of 24 h hyperthermic effect on breast cancer cell line MCF7.

Cell proliferation was measured based on cellular conversion of the colorimetric reagent 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) into soluble formazan by a dehydrogenase enzyme found in metabolically proliferating cells (CellTiter 96 Aqueous One Solution cell proliferation assay; Promega, Madison, WI). Following each treatment, 20 μL of dye solution was added into each well in a 96-well plate and incubated for 2 hours. Absorbance was recorded at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices, Sunnyvale, CA, USA).

Cell proliferation was examined using the cell counting kit-8 (CCK-8) (Multiscience, Hangzhou, China) and 5-ethynyl-2′-deoxyuridine (EdU) (RiboBio, Guangzhou, China) incorporation assays. Cells were seeded in a 96-well culture plate at a density of 1 × 10⁴ cells/well in 100 μL culture medium. For the CCK-8 assay, 10 μL CCK-8 medium was added in each well at 0 h or 24 h after transfection. Then, cells were incubated for 4 h at 37 °C. The absorbance value of each well was detected using an ELISA plate reader (Molecular Devices, San Francisco, CA, USA) at 450 nm. For the EdU assay, 100 μL EdU medium (50 μmol) was added in each well 24 h after transfection, and cells were incubated for 2 h at 37 °C. Then, DNA staining solution and EdU staining solution were added in each well to mark living cells (blue) and the proliferating (red) cells according to the manufacturer’s protocols, respectively. We used a fluorescence microscope to observe the cells at 20× and the ImageJ software to determine cell numbers.

Recombinant Pfs25 or VAR2CSA (1 µg/ml in PBS) was coated on Nunc MaxiSorp plates overnight at 4 °C. Plates were incubated with blocking buffer (1 % BSA) for 1 h at room temperature (RT). Plates were washed three times in between different steps. Before adding the anti-sera for incubation with the capture antigen, IgG levels in individual serum samples were equalized by dilution. This was done based on pre-determined OD₄₉₀ ELISA values. IgG-normalized serum samples were added (50 µl per well) to the ELISA plate in triplicates, and incubated for 1 h at RT. The ELISA wells were subsequently washed three times in PBS with 0.05 % TWEEN 20 and 50 µl of freshly made 8 M Urea was then added to the wells for 5 min (reference plates were incubated with PBS). ELISA plates were then washed three times with PBS (0.05 % TWEEN 20). HRP-conjugated polyclonal goat anti-mouse IgG (A16072, Life Technologies, Denmark) was diluted 1:3000 in blocking buffer and incubated for 1 h. Finally, color reactions were developed for 7 min by adding o-phenylenediamine substrate. The HRP enzymatic reaction was stopped by adding 2.5 M H₂SO₄ and the optical density was measured at 490 nm using an ELISA plate reader (VersaMax Molecular Devices). IgG avidity index values were calculated as the ratio of the mean OD value of urea-treated wells to PBS control wells multiplied by 100.

Purified FoB cells were isolated from C57BL/6-C20-CD45.1 mice congenic for Igh^a and adoptively transferred into B6 or Rag2^–/– mice. Recipients were bled at days 0, 7, 14, 21, and 30 and serum was isolated by centrifugation. Nunc Maxisorb 96-well flat-bottom plates (Thermo Fisher Scientific) were coated with 10 μg/mL F(ab’)₂ fragment goat anti-mouse IgM in sodium bicarbonate buffer and blocked with 2% BSA/PBS. Serum was serially diluted across the plate and incubated overnight at 4°C. For detection, biotin anti-mouse IgM^a (Igh-6^a, BD Pharmingen) and biotin anti-mouse IgM^b (AF6-78, BioLegend) were diluted in blocking buffer and added at 1:3000 for 2 hours at room temperature followed by streptavidin-HRP (BD Pharmingen) at 1:10,000 for 1 hour at room temperature. Wells were developed with 3,3’,5,5’-Tetramethylbenzidine substrate solution and stopped with phosphoric acid. Plates were read on an ELISA plate reader (Molecular Devices). Concentrations were extrapolated from a standard curve generated based off the absorbance of normal mouse serum from a C57BL/6-C20-CD45.1 donor.