Biotek synergy 2 plate reader

pRSF-duet-P18NP was transformed into BL21 competent bacterial cells. After overnight IPTG induction (0.1 mM) at 16°C, the cells were lysed and the NP protein was purified on a HisTrap immobilized metal affinity chromatography (IMAC) column (Cytiva, USA). The Nunc MaxiSorp plates (Thermo-fisher, USA) were coated with 200 ng of recombinant NP proteins overnight at 4°C in coating buffer (50 mM Carbonate-Bicarbonate buffer, pH 9.5). Plates were washed once with wash buffer (PBS + 0.2% Tween20) and blocked for 2 h at ambient temperature with blocking buffer (PBS + 4.0% dry milk) followed by three more washes. Serum samples were serially diluted in diluting buffer (PBS + 0.05% Tween20), added to plates, and incubated for 1 h. Following three washes, samples were incubated with goat anti-guinea pig IgG HRP secondary antibody (Sigma, USA) at 1:10,000 dilution for 1 h. Following four washes, samples were incubated with the TMB substrate (Thermo-fisher, USA) for 15 min in the dark, then a stop solution (0.16M H₂SO₄) was added and optical density (OD) values were read at 450 nm using a Biotek Synergy 2 plate reader (Biotek, USA). The IgG antibody endpoint titer was defined as the highest dilution giving OD₄₅₀ above the cutoff value, which was determined by average plus four times standard deviation of secondary-antibody-only controls.

Fluorescence spectroscopy was employed to monitor HYP loading in MCF7 and MDA-MB-231 cells. Cells were seeded in 96 well plates. The designated groups were treated with 100 μM BSO overnight. Subsequently cells were treated with vehicle, HYP, BSO+HYP, NAC+HYP and BSO+NAC+HYP for 4 h. All cells were consequently washed twice and all cell groups were placed in complete media. The fluorescence was read in a Biotek synergy 2 platereader (BioTek Instruments, Inc.) using a 530±25 nm bandpass excitation filter and a 590±35 nm bandpass emission filter. Empty wells incubated with HYP and washed twice were used as blanks and subtracted from all data groups.