Spectramax paradigm plate reader

To assess the levels of cytokines in the plasma or conditioned media of cultured PBMCs, enzyme-linked immunosorbent assay (ELISA) test kits for IL-1β and TNF-α (R&D Systems) were used^{19 (link)}. The absorbance of triplicate samples was read by SpectraMax Paradigm Plate Reader (Molecular Devices).

The affinity of a protein-DNA interaction can be determined by monitoring the changes in FP associated with the formation of the complex under equilibrium conditions [67 (link)]. We used FP to study the equilibrium binding between the Sso7c4 proteins (wild-type, C-terminally truncated, Sso7c4-R11A, Sso7c4-R22A and Sso7c4-R11A/R22A) and 5′ FAM-labeled dsDNAs of different lengths. In a basic FP experiment, the protein was serially diluted in binding buffer (each sample with a volume of 50 μl) in the wells of a 96-well microplate (NUNC). Then, the 5′ FAM-labeled dsDNA was added to each well (50 μl of a 200 nM stock) to a final DNA concentration of 100 nM and a total binding reaction volume of 100 μl. In the binding assay using the Sso7c4-R11A/R22A mutant and 20-bp dsDNA, the mutant protein was serially diluted from a higher concentration of 4.33 mM to confirm its much weaker binding affinity. The binding reactions were incubated at room temperature for 1 h. After the binding reaction had reached equilibrium, triplicate reactions for each sample were measured at 25°C using the SpectraMax Paradigm plate reader (Molecular Devices, CA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The equilibrium binding data were analyzed and plotted using Origin Pro8.

ELISA was used to measure binding of H1ssF WT, H1ssF gN38, and H1ssF R38 nanoparticles to mAbs CR6261, CR8020, MEDI8552, FI6v3, C179, 02-1H01, and D25 (anti-RSV). 96-well plates were coated with 2 μg ml⁻¹ H1ssF WT, H1ssF gN38, and H1ssF R38 (0.1 ml per well) and incubated at 4 °C overnight. For testing HA-specific IgG levels in immune sera, plates were coated with 2 μg ml⁻¹ of recombinant HA proteins derived from H1 NC99 WT, H1 NC99 gN38, H5 IN05, and H7 SH13. Plates were then blocked with PBS containing 5% skim milk at 37 °C for 1 h. Monoclonal antibodies and immune sera were serially diluted in four-fold steps and added to the wells for an hour. Horseradish peroxidase (HRP)-conjugated anti-human or anti-mouse IgG (Southern Biotech) was added and incubated at 37 °C for 1 h. The wells were developed with 3,3′,5′,5-tetramethylbenzidine (TMB) substrate (KPL), and the reactions were stopped by adding 1 M H₂SO₄ before measuring absorbance at 450 nm with a Spectramax Paradigm plate reader (Molecular Devices).

β-OHB levels in mouse serum, HepG2 or AML12 growth medium were determined using a spectrophotometric enzymatic assay (MAK134, Sigma) according to the manufacturer’s protocol. Total protein was also isolated from each well and used to normalize β-OHB levels in the growth medium of HepG2 cells. To remove interfering substances, growth medium samples were filtered with Amicon Ultra-0.5 Centrifugal Filter Units (UFC501096) prior to measurement. The optical density was assessed on a SpectraMax Paradigm plate reader (Molecular Devices) at 340 nm.

Blood samples were collected in commercial gel tubes using BD Vacutainer® SST II plastic serum tubes with silica (clot activator) gel (Becton-Dickinson, Plymouth, UK). Sera were separated by centrifugation (4°C, 2000g, 10 min) and kept at -20°C until assayed. cfDNA was quantified on coded serum samples, by a rapid SYBR^® Gold fluorometric assay, which does not require prior processing of samples, i.e., DNA extraction and amplification [12 (link)]. Briefly, 20μL of patient serum was applied in duplicate to black 96-well plates (Greiner Bio-One; Frickenhausen, Germany). 80μL of diluted SYBR® Gold (Invitrogen, Paisley, UK) was added to each well (final dilution 1:10,000), and fluorescence was measured with a 96-well fluorimeter (SpectraMax Paradigm plate reader, Molecular Devices, San Jose, CA) at an emission wavelength of 535 nm and an excitation wavelength of 485 nm. Concentrations of unknown samples were calculated by extrapolation in a linear regression model, from a standard curve (39–5000 ng/ml) of sonicated Salmon sperm DNA (Sigma-Aldrich).