Spd121p speedvac concentrator

The extraction of the polyphenols was performed as described by Abountiolas et al. [31 (link)] with minor adjustments, such as using the fruit’s puree instead of juice. Triplicates of 5 g of homogenate were mixed with 15 mL of acetone and homogenized using a polytron for 1 min. Samples were sonicated for 10 min, and finally filtered through Whatman paper No. 4. The filtrate was concentrated to 5 mL in an SPD121P SpeedVac^® Concentrator (Thermo Fisher Scientific Inc., Asheville, NC, USA) and finally passed through a classic C18 Sep-Pack cartridge (Waters Technologies Corp., Milford, MA, USA). Before passing the concentrated sample, the Sep-Pack cartridge was activated with ~5 mL of methanol, followed by ultrapure water and 3% acidified water. Anthocyanins and other phenolic compounds were absorbed into the cartridge, while sugars, acids, and other water-soluble compounds were eluted with ~10 mL of acidified water. The phenolic compounds were then recovered by passing ~2 mL of methanol (containing 3% formic acid) through the cartridge. The extracted sample was filtered through a 0.20 µm syringe filter into 2 mL autosampler vials and stored at −30 °C until analysis.

Brain samples were weighed and homogenized using a Bertin Precellys 24 Tissue Homogenizer in a screw-top microcentrifuge tube containing ∼0.1 ml of zirconia/silica beads (1 mm, BioSpec Products) and 1.0 ml of LC-MS grade isopropanol:water:ethyl acetate (30:10:60, v:v:v) containing SPLASH Lipidomix Mass Spec Standard (Avanti Polar Lipids). This was followed by sonication for 5 min and then centrifugation for 5 min at 15,000g at 4 °C. Supernatants were transferred to new tubes and brought to dryness using a Thermo Savant SPD121P Speedvac Concentrator. They were then reconstituted in 0.5 ml of LC-MS grade isopropanol:acetonitrile:water (45:35:20, v:v:v), vortexed briefly, and sonicated for 5 min. Finally, samples were centrifuged for 10 min at 15,000g at 4 °C and supernatant was transferred to auto-sampler vials.

HASMCs (8 × 10⁵ cells) were seeded on 60 - mm tissue culture dishes (245389, Nunc). After 24 hr, rapalogues in ethanol were treated for 2 hr to the cells at final concentrations of 2 μM or 10 μM in 2.5 ml culture media. The cells washed with PBS quickly and extracted with 1 ml methanol for 2 hr at −20 °C. After centrifugation at 12,000 × g for 10 min, Supernatants were dried using a savant SPD121P Speedvac Concentrator (Thermo Fisher Scientific), dissolved in the 200 μl of 50% Acetonitrile with 0.1% trifluoroacetic acid solution. Samples were separated by a reverse-phase high performance liquid chromatography (Agilent Series 1100 HPLC system with diode array). 100 μl of each samples were injected to a C₁₈ column (4.6 × 250 mm ID, 5 μm spherical packing; Vydac) and eluted through 50–95% Acetonitrile with 0.1% trifluoroacetic acid mobile phase solution at a rate of 1.2 ml/min. Amounts of extracted drugs from cells were calculated by using each standard curves gained from standard drug solutions (1.25, 2.5, 5, 10, 20 μM). Drugs were detected by UV absorbance at 278 nm.