Hypersil bds c18 column

CD4⁺ T cells isolated from the injured lungs 7 d post induction of ALI were sorted into GIBCO Hank's Balanced Salt Solution (HBSS) using FACS. After two washing steps with HBSS 250.000 cells were resuspended in 350 µl HBSS. The nucleotides ATP, AMP, cAMP and ADP-ribose (ADPR) were then added with a final concentration of 20 µM and incubated at 37 °C. To stop the reaction at indicated time points (2, 4, 12, 30 and 60 min) an aliquot of 60 µl of the incubation medium was transferred into an Eppendorf tube on ice and centrifuged at 4 °C for 2.5 min at 550 g. Aliquots of 30 µl were applied to a reverse phase HPLC system using a Hypersil BDS C18 column (Thermo Fisher Scientific, Langerwehe, Germany) as previously described [23] (link). Briefly, purines were separated by running a linear gradient of buffer A (150 mM potassium dihydrogen orthophosphate) and buffer B (15% acetonitrile in buffer A) using a low pressure gradient mixing device. The absorbance was measured at 254 nm and the retention times were assessed using standard samples of nucleotides and nucleosides.

Reconstituted supernatants (10 µl) were injected into a Capcell Pak C8 HPLC column (50 × 2.1 mm inner diameter; Phenomenex Inc., Torrance, CA) at 45°C or a Hypersil BDS C18 column (30 × 2.1 mm inner diameter, 3 µm particle size; Thermo Fisher Scientific), each coupled to a tandem mass spectrometer (API4000; AB SCIEX LLC). The mobile phase was 25 mM ammonium formate buffer, pH 3.5. Compounds were eluted with a linear gradient at a flow rate of 250 µl/min (Capcell Pak C8 column) or 300 µl/min (Hypersil BDS C18 column). Eluted compounds were ionized using an electrospray interface.
Drug concentrations in brain homogenate were quantified against calibration curves generated by spiking the drugs into drug-free brain homogenate. Average values for the triplicate measurements were calculated and considered as n = 1. Summary statistics were calculated using Microsoft Office Excel, 2003 (Microsoft, Redmond, WA).

HPLC separation and quantification of phenolic compounds in apple extracts and samples obtained from in vitro digestion, were performed according to earlier studies [23 (link)] with some modifications. Identification was possible by comparing spectra and retention times with those of commercial standards and with those reported in previous works [29 (link)]. The column selected was a Hypersil BDS C18 column (250 mm, 4.6 mm, 5 μm) (Thermo, Bellefonte, PA, USA). Analyses were run on a Finnigan HPLC system (Thermo Electron Corporation, San Jose, CA, USA) provided with a photodiode array detector (DAD). The identity of phenolic acids and flavonoids was confirmed with LC-ESI/MS/MS experiments, as already reported [24 (link)].

All samples were processed and analyzed as previously described (Lyte et al., 2022 (link)). Briefly, thawed samples were homogenized in a BeadRuptor and then centrifuged at 3,000 × g and 4°C for 15 min. Yolk samples required heating to 37°C and diluting 1:10 with mobile phase in order to pass through the spin filters. Sample supernatant was passed through 2–3 kDa spin filters, and flow-through was stored at −80°C until ultra-high performance liquid chromatography with electrochemical detection (UHPLC-ECD). The UHPLC-ECD consisted of a Dionex Ultimate 3,000 autosampler, a Dionex Ultimate 3,000 pump, and Dionex Ultimate 3,000 RS electrochemical detector (ThermoFisher-Scientific, Sunnyvale, CA). Mobile phase was buffered 10% acetonitrile (Catalog #: NC9777698, ThermoFisher-Scientific) and the flow rate was 0.6 mL/min on a 150 mm (length) 3 mm (internal diameter) 3 µm (particle size) Hypersil BDS C18 column (Catalog #: 28,103-153030, ThermoFisher-Scientific). A 6041RS glassy carbon electrode set at 400 mV was used for electrochemical detection. Data were analyzed using the Chromeleon software package (version 7.2, ThermoFisher-Scientific), and neurochemical identification was confirmed using the retention time of the corresponding analytical standard (Millipore-Sigma, St. Louis, MO) (for norepinephrine, Catalog #: 636-88-4; for L-dopa, Catalog #: 59-92-7).

Cell optical density (OD) was observed at 600 nm using a TU-1810 spectrophotometer. The fermentation broth was sampled by directly centrifuged, then the supernatant was filtered and analyzed by a Hitachi Primaide HPLC system (Japan). 3-Phenylpropanol, cinnamic acid, 3-phenylpropionic acid, 3-phenylpropyl aldehyde, cinnamyl alcohol and l-phenylalanine were separated by a Thermo Scientific Hypersil BDS C18 column (150 × 4.6 mm, 5 μm) and measured by a PDA detector at 210 nm with a mobile phase (20% acetonitrile, 80% water, 0.1% trifluoroacetic acid) at 1 mL/min. Glucose and glycerol were measured by a Morphling™ Sugar-H column (300 × 7.8 mm, 5 μm) and a RI detector with a mobile phase (5 mM H₂SO₄) at 0.6 mL/min, 65 °C. All of aforementioned compounds were quantified by HPLC analysis using a five-point calibration curve with the R² coefficient higher than 0.99. GC–MS analysis for 3-phenylpropanol identity was conducted by Agilent Technologies gas chromatography-triple quadrupole tendem mass spectrometry 7890B-7000D. The oven temperature was initially held at 50 °C for 4 min. Next the temperature was increased at 5 °C/min to 150 °C and then at 90 °C/min to 250 °C. Temperatures of the injection port and the ionizing source were 250 °C and 280 °C, respectively. The split ratio was 10:1 and 1 µL of sample was injected.

Immediately following dissection, brain tissue samples were preserved in 0.2 M HCLO4 and stored at −80°C until UHPLC preparation. Brain regions were homogenized using the Omni Bead Ruptor system. Sample homogenates were then centrifuged for 30 min at 3,000 g. An Ultra-High-Performance Liquid Chromatography with electrochemical detection (UHPLC-ECD) system was used to determine neurochemical and metabolite concentrations as described previously (Villageliú et al., 2018 (link)). A Dionex Ultimate 3000 autosampler system queued samples before injection. Separation of neurochemical target compounds was completed via a 150 mm long Hypersil BDS C18 column (Thermo Scientific, Sunnyvale, CA, USA) using 0.6 ml min⁻¹ flow rate of 10% buffered acetonitrile MD-TM mobile phase (Thermo Scientific, Sunnyvale, CA, USA). The column used had a diameter of 3 mm. Particle and pore size was 3 μm and 130 Å, respectively. Electrochemical detection was achieved with 6041RS glassy carbon electrode at 400 mV with a limit of detection set at 5× the signal-to-noise ratio. Neurochemical standards were purchased from Sigma–Aldrich (St. Louis, MO, USA).