Dps 20 dual processing system

The 4 mM ouabain stock solution was prepared and dissolved in DMSO. Ouabain was diluted to 20 μM in normal saline and 20 μL of the diluted ouabain or DMSO (vehicle control) was added to the primary cell cultures apically and incubated for 2 h in 37°C supplied with 5% CO₂. Primary cultured epithelial cells were washed apically with 100 μL of PBS 24 h before the experiment. A total of 10 μL of the apical fluid was immediately added onto pH test strips. After pH reading, all remaining apical fluid was removed, and another 20 μL of 20 μM ouabain or DMSO was added with 50 μL of PAO1 suspended in normal saline (10⁷ CFU/insert). Cells were then incubated for additional 5 h for biofilm to form. All apical supernatant was collected for determining the CFU of unattached planktonic bacteria. Biotic biofilm assay was used by counting CFU of bacterial biofilm formed on the epithelial cells. ALI membrane was removed from the filter and sonicated in 2 mL of PBS for 30 s at 80% amplitude by the DPS-20 dual processing system (130 W; PRO Scientific) to disassociate congregated PAO1 biofilm. After sonication, both planktonic and biofilm samples of PAO1 were plated on tryptic soy agar plates for total CFU counting.

Pentobarbital and pentobarbital-d5 certified reference material (CRM) in methanol were purchased from Cayman chemicals (Ann Arbor, MI). Pentobarbital (PB) plasma and brain concentrations were determined using a UHPLC-MS/MS analytical method with the same instrumentation as the midazolam method. Pre-weighed brain samples were homogenized using 4 volumes of LC–MS grade water on a DPS-20 dual processing system from Pro scientific Inc. Sample preparation included protein precipitation with acetonitrile in the presence of pentobarbital-d5 internal standard (IS). Chromatographic separation was achieved using a Phenomenex Kinetex XB-C18 analytical column (2.6 µm, 100 Å, 2.1 × 50 mm) at 40 ± 2 °C. Gradient elution was employed where mobile phase A (MP-A) was 0.1% formic acid in water and mobile phase B (MP-B) was 0.1% formic acid in methanol. A flow rate of 0.4 mL/min and a 5 µL injection volume were used. Detection of PB and IS was carried out in the positive ion mode utilizing electrospray ionization (ESI) MRM, by monitoring the precursor to product ion transition pairs of 225.1–182.1 m/z for PB, and 230.1–187.1 for IS. The analytical data obtained were processed using Sciex Analyst software™ (version 1.7.1).