Up50h ultrasonic processor

Prior to exposure experiments, 0.9 mg of dried and extracted PM2.5, as described above, was dissolved in 900 μl of cell media (CM; supplemented RPMI-1640 medium, see below) in 1.5 ml Eppendorf tubes and subjected to indirect and direct sonication. Indirect ultra sonication was performed at 4°C and at 120 W for 15 min using an Ultrasonic Cleaner water bath (Mettler Electronics), followed by direct sonication at room temperature (RT) at 50 W, 0.05 cycle, 20% amplitude for 60 s using an UP50H Ultrasonic Processor (Hielscher Ultrasound Technology). The immersion was aliquoted and diluted to desired concentration. The CM without PM, used on control cells, underwent the same protocol and maintained the same volumes. The direct sonication step followed by vortex was performed prior to each exposure.

The whole hippocampus was lysed in cold RIPA buffer (50mM TRIS, 1% IGEPAL, 50 mM EDTA, 150 mM NaCl, 0.05% SDS, 1% Triton X-100, pH 7.4), supplemented with proteases and phosphatases inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Samples were homogenized with a UP50H ultrasonic processor (Hielscher Ultrasonics GmbH, Teltow, Germany) and lysates centrifuged at 13.000 rpm for 10 min. Proteins were separated by electrophoresis with 8% SDS-PAGE gels for 3 h at 90V and subsequently transferred to a nitrocellulose membrane. Proteins were visualized by ECL (Thermo Scientific) and pictures were obtained by ChemiScope 3200 mini (Clinx Science, Shanghai, China). The antibodies used are listed in Table 2.

The 200 µL of iced cold Sterile PBS was added to the samples (besides samples containing PTX 20 µM in the medium). These were homogenized with the Ultrasonic Processor UP50H (Hielscher): 1 cycle—60% amplitude. The run consisted of two cycles of 15 s with a rest of 50 s in ice for each sample. The samples for assessing the bioavailability of VNA and PTX and amino acids and neurotransmitters quantification were prepared the same way. To 100 µL of homogenate 200 µL of the internal standard (IS) Rolipram (Sigma Aldrich, Buchs, Switzerland) (20 ng/mL) for the positive MS experiment, and VPA d-6 (Sigma Aldrich, Buchs, Switzerland) (50 µg/mL) for the negative MS experiment were added. Samples were vortexed for 10 s and then centrifuged at 12,000 rpm for 10 min at 4 °C. After centrifugation, 150 µL of supernatant was transferred into LC-MS vials already containing 120 µL of MilliQ water. Then, 10 µL was injected into the LC-MS/MS system.

For the analyses of metabolites and enzyme activities, 30 mg of powdered mucosa tissue was homogenized in 300 µl lysis puffer containing 10 mM HEPES (Thermo Fisher Scientific, Schwerte, Germany), 1% (v/v) Tween20 (Carl Roth, Karlsruhe, Germany), 1 mM EDTA (GE Healthcare, Munich, Germany), 10 mM NaF (Thermo Fisher Scientific), 0.1% (v/v) Triton X-100 (GE Healthcare), 0.5% (v/v) DOC (Sigma-Aldrich), 0.1% (w/v) SDS (USB Corporation, Cleveland, OH, USA) with 0.5 cycles and 80% amplitude (20-times) Ultrasonic Processor UP50H (Hielscher Ultrasound Technology, Teltow, Germany)^{54 (link)}. The homogenized extract was centrifuged at 3000×g for 20 min at 4 °C. The supernatant was used to measure glucose and lactate concentrations and aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) activities photometrically (Abx Pentra 400; Horiba, Kyoto, Japan) using kits for glucose (no. A11A01667, Axon Lab, Reichenbach, Germany), lactate (no. A11A01721, Axon Lab), AST activity (no. A11A01629, Axon Lab) and GLDH activity (LT-GD 0010, Labor + Technik Eberhard Lehmann GmbH, Berlin, Germany). Protein concentrations of the extracts were measured using the Bradford kit (Thermo Fisher Scientific). Metabolites and enzyme activities were normalized to the protein concentration of the mucosa extract.

The total protein extract was obtained from 0.1 g of mouse liver tissue (previously stored at −80 °C) homogenized in 1 mL of 0.1 M Tris/5 mM EDTA buffer, pH 7.4 and sonicated on ice 3 times for 30 s, using an Ultrasonic Processor UP50H from Hielscher (Hielscher Ultrasound Technology, Teltow, Germany) set at 1 cycle and 80% amplitude. After 1 h of incubation at 4 °C, the tissue homogenates were centrifuged at 10,000 rpm and 4 °C for 30 min. In the end, the supernatant was collected for further biochemical determinations. The total protein concentration was measured using Lowry’s method (1951) and bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) as standard [55 (link)].