hIL-10 protein concentrations were determined in supernatants from TLR9-stimulated cultures by standard capture ELISA (Ready-SET-Go, eBioscience, Vienna, Austria) according to the manufacturer's instructions Plates were read at 450 nm once the substrate had developed, and cytokine concentration was determined by extrapolation from the standard curve.
Quantification of DNA was performed using Promega QuantiFluor dsDNA System (Madison, WI), as per the manufacturer's multiwell plate instruction and plates read on a Thermo Scientific Varioskan Flash (Rockford, IL).
After you fight through the scrum to put your name on a list, finally receive your "meal ticket" (on which countermen mark your order), get to your seat, and take in the instructions — no plates, cocktail service only, a fifty-buck fine if you lose your ticket — you stand in line for meat, and again for sides, and once more for drinks.
The Taqman® microRNA reverse transcription kit (Applied Biosystems, Warrington, UK) and Taqman® microRNA assays (Applied Biosystems) were used according to the manufacturer's instructions and plates were read using an ABI Prism 7900 Real-Time PCR system (Applied Biosystems).
The two commercial ELISAs for the diagnosis of JEV were performed in Vientiane following the manufacturer's instructions, with plates read at 450 nm using a Multiskan EX ELISA plate reader (Labsystems, Franklin, MA).
After 48 h of incubation at 37 °C, caspase-3/7 substrates (Promega) were added according to the manufacturer's instructions and plates were incubated for 1 h at 37 °C in the dark.
Activity assay was performed using a Caspase-Glo 3/7 assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and plates were incubated for 1 h at room temperature before reading on a luminometer.
Supernatants yielding extracellular bacteria were collected, diluted according to protocol instructions, and plated on P. aeruginosa isolation agar (BD Diagnostics Sparks, MD, USA).
Half of the ligation reaction (10 μL) was transformed into XL1-Blue competent E. coli cells (Agilent) according to manufacturer's instructions and plated on LB-agar containing 100 μg/mL ampicillin.
To prepare samples for western blotting, protein concentrations were determined using the PierceTM BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions for plate reader.
