Fluostar omega multi mode microplate reader

MEF‐β6 cells were plated in 10‐cm petri dishes and incubated at 37°C until 80% confluence. Inhibitors were added along with fresh medium, containing 10% FCS and 2 ng mL⁻¹ TGF‐β. After 4 h of incubation at 37°C, nuclear protein was prepared using Nuclear Extract kit (Active Motif, La Hulpe, Belgium). Protein concentration was determined using BCA Protein Assay kit (Pierce, Rockland, IL). A quantity of 10 μg of nuclear protein was loaded into a Pathscan pSmad2 ELISA assay (Cell Signalling, Danvers, MA). Absorbance was read at 450 nm using a FLUOstar omega multi‐mode microplate reader (BMG Labtech).

Reactive oxygen species (ROS) detection was undertaken using the fluorescent 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFDA) assay (Sigma-Aldrich). Cells were seeded at 1 × 10⁵ and incubated for 24 h. On the day of treatment, the test chemical and positive control were diluted accordingly. The 4000× DCFDA stock was diluted to 1× DCFDA, and 100 µl was added to each well in a 96-well plate. The cell flasks were treated with CdCl₂, and a 100 µl aliquot was immediately added to the 96-well plate. Fluorescence readings were taken using a FLUOstar Omega Multimode microplate reader (BMG LABTECH Ltd, UK) with excitation/emission set at 485/535 at various time points from the time of dosing; 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h. Hydrogen peroxide (50 μM) was used as a positive control in all experiments.

A549 cells were seeded at 10,000 cells/well in 96-well plates and then co-transfected with 25 ng empty or pmirGLO-NRAS 3′UTR and 175 ng empty or pmR-ZsGreen1-let-7a-1 in varying ratios. After verifying high efficiency at 24 h post transfection, the Dual-Luciferase Reporter Assay System (Promega) was done following the manufacturer’s instructions. Briefly, cells were washed with PBS and lysed with 20 µl/well 1× Passive Lysis Buffer for 20 min with shaking at 60 rpm. Debris was spun down at 2500 g for 5 min, and then 5 µl/well of lysate was transferred to an opaque white plate. The FLUOstar Omega multi-mode microplate reader (BMG Labtech) was used for luminescence measurements according to the following protocol: per well, an initial injection of 100 µl Luciferase Assay Reagent, 10 s measurement of luminescence, followed by a second injection of 100 µl Stop & Glo Reagent, and another 10 s measurement, detecting the firefly and Renilla luciferase signals in sequence for each well. For normalization, the firefly luciferase signal from each well was divided by the corresponding Renilla signal. The same assay was also done co-transfecting A549 cells with empty or pmirGLO-NRAS 3′UTR and si-control or si-circPVT1.

Bacterial DNA was prepared from E. coli–competent cells (Thermo Fisher Scientific) using DNeasy Blood and Tissue Kit (Qiagen). PBMCs freshly isolated from each study participant (5 × 10⁵ cells) were cocultured with E. coli bacterial DNA (0 to 3 × 10⁴ DNA copies [7.5 pg/mL]). Coculture supernatants were collected and analyzed on day 3. IFN-γ levels in the supernatants were measured using a human IFN-γ ELISA (R&D Systems) and the FLUOstar Omega multimode microplate reader (BMG Labtech). For bacterial DNA digestion, E. coli bacterial DNA was incubated with DNase I (Thermo Fisher Scientific) at 37°C for 15 minutes. For BTK inhibition, PCI 29732 (final concentration 0.5 nM, Tocris Bioscience) (42 (link)) was added to PBMC cultures at 2, 8, and 12 hours prior to the addition of E. coli bacterial DNA (3 × 10⁴ copies, 7.5 pg/mL). To examine potential interference by human cell-free DNA, an equivalent concentration (7.5 pg/mL) of human genomic DNA (Promega) was added to the bacterial DNA and PBMC coculture, and IFN-γ output was measured as described previously.

Concentrations of sCD14, LBP, zonulin, and I-FABP were determined in serum (with dilutions of 1:2500, 1:1000, 1:10, 1:10, respectively) using separate immunoassays (R&D Systems). Serum IFN-γ, diluted 1:2, from patients not on a systemic immunosuppressant was determined using a human IFN-γ ELISA (R&D Systems). FLUOstar Omega multimode microplate reader (BMG Labtech) was employed. Representative data from duplicate experiments were presented.