Polarstar optima plate reader

Superoxide production in aortic rings was measured using L-012 chemiluminescence. The assay is based on methods developed by Miller et al. (14 (link)) with the following modifications. Aortic segments were cleared of fat and connective tissue and cut into 2–3 mm long segments, which were incubated at 37°C for 30 min in Krebs-HEPES buffer either alone or in the presence of apocynin (300 μM), a non-specific NADPH oxidase (Nox) inhibitor, in a cell culture plate. Krebs-HEPES buffer (300 mL), containing L-012 (100 mM, Wako Pure Chemicals, Osaka, Japan) and the appropriate treatments were placed in a 96-well optiplate, which was loaded into a Polarstar Optima plate reader (BMG Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37°C. After background reading was completed, a single aortic segment was added to each well in the optiplate and photon emission was recounted. Superoxide production was quantified by subtracting the final reading from the background reading, and counts were then taken as arbitrary units of superoxide production and expressed as a ratio to dry tissue mass (AU/mg dry tissue).

For FP, 6 µM of FITC-labeled α1 peptide and 4 µM of FITC-labeled β2 peptide were incubated with varying concentrations of A10 (0–35 µM), A7 (0–35 µM), G8 (0–35 µM) or G11 (0–10 µM) scFvs and A10 scFv-Fc (0–1 µM), respectively, in elution buffer (125 µL; PBS+200 mM imidazole) overnight at 4°C or 2 h at RT. The resulting complexes were transferred to Corning® 96 well half-area, polystyrene black plates (Corning, NY), and FP values were measured using the POLARstar OPTIMA plate reader from BMG LABTECH (BMG LABTECH Inc., Cary, NC). The resulting millipolarization (mP) values were plotted against concentration and dissociation constants calculated using OriginPro8.5 software (OriginLab Corp., Northampton, MA).

The luciferase reporter assay was performed by using Lipofectamine 3000 reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. The mouse, equine, and human cells were seeded at 50% confluency in 24-well plates, treated with 0 or 20 ng/mL (0.5 mL/well) of murine (Cell Sciences, Canton, MD, USA), equine (R&D Systems, Minneapolis, MN, USA), or human IFN-γ (Cell Sciences), respectively, and cotransfected with 0.07 pmol of reporter vector and 0.07 pmol of effectors in each well at 24 h post-treatment. Four microliters of Lipofectamine 3000 were diluted with 114 μL of Opti-MEM medium (Invitrogen, San Diego, CA, USA). DNA and 1.5 μL of P3000 reagent were mixed with 114 μL of Opti-MEM medium. The total amount of DNA was adjusted to the same amount with pSVSPORT1 DNA. The solutions were combined and incubated at room temperature for 15 min, and one-third volume was transferred into each of three wells of the four human cells. At 40 h post-transfection, luciferase activity was measured with a luciferase assay kit (Promega, Madison, WI, USA) and a Polarstar Optima plate reader (BMG LABTECH Inc., Cary, NC, USA).

Treatment of cells with Etoposide was carried out by plating EGFP, SIAE and SIAE-S127A expressing clones at 2,500 cells per well in 100 μL clear media in 96 well plates. Cells were plated in the presence and absence of doxycycline. After 48 of doxycycline treatment cells were treated with Etoposide (Sigma) prepared in dimethylsulfoxide (DMSO; Sigma). Concentrations ranged from 1 μM to 14 μM in 1 μM increases. Untreated cells and vehicle controls were included. Cells were treated for 72 hours post-chemotherapy addition. Phase-contrast images were taken and MTS assays using CellTiter 96 AQ_ueous One Solution (Promega) were carried out at the end of the time course according to manufacturer’s instructions. Plates were incubated at 37 °C for 3 hours. The absorbance was then read at 490 nm using the BMG labtech POLARstar Optima plate reader.

Triplicates of scaffolds seeded with osteoblasts were cultured with osteogenic media in 15 mL Falcon tubes for 1, 10, 20, 30 or 40 days at 37 °C, 5%CO₂. At each termination point, scaffolds were washed twice with PBS, transferred to 1.7 mL Eppendorf^® tubes (Eppendorf, Macquarie Park, Australia), centrifuged at 500 rpm for 2 min at room temperature to remove excess liquid and stored at −80 °C until analysis. For analysis, scaffolds were digested with 50 µg/mL proteinase K (Sigma Aldrich) in 1× TE at 50 °C for 48 h. DNA content for 100 µL of each sample in triplicate were measured and quantified using a Quant-iT^™ PicoGreen^® dsDNA assay kit (Thermo Fisher Scientific, Scoresby, Australia) according to the protocol supplied by the manufacturer (Invitrogen). An equal volume of the aqueous Quant-iT^™ PicoGreen^® working solution was added to each triplicate. After three minute incubation on a rocking plate, fluorescence was measured at λ_excitation = 485 nm and λ_emission = 520 nm using a POLARStar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany).

The kinase assay was performed with a Beacon Tyrosine kinase assay kit (Invitrogen, A-35725) according to the manufacturer’s instructions. The kinase buffer was prepared by supplementing the provided buffer with dithiothreitol (DTT) (Final concentration: 2 mM) and dimethyl sulfoxide (DMSO) (Final concentration: 0.1%). In the kinase buffer, Dasatinib (Final concentration: 10 μM; Selleck Chemicals; Houston, TX, # S1021), 2H7 and wild-type FN3 (WT-FN3) (Final concentration for both: 7.5 μM) were mixed with Lyn kinase (final concentration: 105 nM; Invitrogen, # P2907). The mixture was incubated at 4°C for 30 min and then at room temperature for 20 min. The detection complex was prepared by mixing the Oregon green, an anti-phosphopeptide antibody and a peptide substrate. As a positive control (highest fluorescence signal), the anti-phosphopeptide antibody was excluded in the reaction, as a negative control (lowest fluorescence signal), the recombinant Lyn kinase was excluded in the reaction. The detection complex was added to the mixtures prepared above, followed by 10 min incubation at 30°C and then the addition of ATP. After another 12 min incubation at 30°C, each reaction was loaded into three adjacent wells of a non-binding microtiter plate (Corning; New York, NY, # 3915) and scanned by POLARstar OPTIMA plate reader (BMG LABTECH).

NHU cell cultures were seeded at 1.5 × 10⁵ cells/well in 96-well plates (Corning Primaria, Fisher Scientific UK Ltd.), and 24 hours after seeding were treated with ketamine in replicates of six. After 72 hours, apoptosis was assessed with the Sensolyte active caspase 3/7 kit used according to the manufacturer's instructions (Anaspec; supplied by Cambridge Bioscience, Cambridge, UK). Briefly, cells were lysed and any active capase-3/7 cleaved a quencher from the AFC (7-amido-4-trifluoromethylcoumarin) fluorophore to generate bright blue fluorescence. Caspase activity–associated fluorescence was measured on a PolarStar Optima plate reader (BMG LabTech) at excitation and emission wavelengths of 355 and 492 nm, respectively.