Neuron culture medium was replaced by Hanks' balanced saline solution buffer with the addition of 56 mM KCl and incubated for 15 minutes at 37°C. The medium was collected and centrifuged to clear cell debris. Neuron pellet was also collected. Samples were immediately frozen in liquid nitrogen and stored at −80°C. For HPLC analysis, samples were thawed and concentrated using a vacuum (Savant SDP 121P, Thermo Fisher Scientific) connected with refrigerated vapor trap (Savant RVT 5105, Thermo Fisher Scientific), and the freeze‐dried samples were resuspended in 10 mM perchloric acid. Monoamines were analyzed by HPLC electrochemical detection by dual channel Coulochem III electrochemical detector (model 5300; ESA Inc., Chelmsford, MA, http://www.esainc.com ), and monoamines were separated by using a reverse phase C18 column (3‐mm 3 150‐mm C‐18 RP‐column; Acclaim Polar Advantage II; Thermo Fisher Scientific) with a flow rate of 0.600 ml/minute. Monoamine concentrations were quantified by comparison of the area under the curve to known standard dilutions.
Savant rvt5105
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Savant RVT5105 is a refrigerated benchtop vacuum concentrator designed for the concentration and drying of samples. It features a temperature range of -20°C to 80°C and can accommodate sample volumes up to 5 liters.
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Lab products found in correlation
Savant sdp121 p, by Thermo Fisher Scientific (1 mentions)
Acclaim polar advantage 2, by Thermo Fisher Scientific (1 mentions)
Savant sc250exp, by Thermo Fisher Scientific (1 mentions)
Tem 1400 electron microscope, by JEOL (1 mentions)
Quick start bradford protein assay kit, by Bio-Rad (1 mentions)
Savant spd131dda speedvac concentrator, by Thermo Fisher Scientific (1 mentions)
5 protocols using savant rvt5105
1
Quantifying Neuronal Monoamine Levels
2
Quantification of Cytokine Levels in BAL
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To quantify the levels of cytokines, BAL was concentrated 50 times using Thermo Scientific Savant RVT5105 Refrigerated Vapor Trap. In this vacuum concentrator, samples are held in a rotor that spins at 1700 RPM creating a centrifugal force, and the protein is dried by evaporation; protein concentration was equated to 200 µg/mL for each sample, and a commercially available ProcartaPlex Human TH1 TH2 CYTO PANEL 11PLEX Th1/Th2 panel (Thermo Fisher Scientific, Waltham, MA, USA, cat. EPX110-10810-901) was used according to the manufacturer’s instructions. Interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18, interferon (IFN)-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α were quantified. Multiplex assays were performed according to the manufacturer’s instructions. Samples were homogenized and adjusted for further quantification in the Luminex®LABScan 100 (Luminex Corp. Austin, TX, USA) system. The xPONENT 3.1 software (Luminex Corp. Austin, TX, USA) was used for data acquisition and analysis. Cytokine concentration was calculated from the standard curve using a five-parameter logistic curve fitting, and cytokine levels below the standard range were extrapolated to give approximate values.
3
Ultrasound-Assisted Methanol Extraction
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Ultrasound-assisted methanol extraction was carried out in triplicate in 4 mL amber glass vials. Masses of 100 mg of the ŌKU commercial sample, and the Arapaoa and Pōhara fresh samples were ground as above and extracted with 2 mL of 80% MeOH ultrasonication for 30 min in an ultrasonic bath (Soniclean 80T, Soniclean Pty Ltd., Adelaide, Australia) on the highest power setting. The extract was centrifuged at 5000× g for 5 min, and 1.5 mL of supernatant was collected. Another 1.5 mL of 80% methanol was added, and the pellet was re-extracted and centrifuged. This re-extraction was repeated until three volumes of supernatant were combined (4.5 mL). Extracts were dried in a SpeedVac centrifugal concentrator (Savant SC250EXP; Thermo Scientific, Hampton, VA, USA) coupled with refrigerated vapour trap (Savant RVT5105, Thermo Scientific, Hampton, VA, USA) and the dry residue massed and resuspended in 10 mL 80% MeOH. A fourth extraction volume was obtained and analysed directly to assess extraction completeness.
4
Albumin-Triclosan Formulation Preparation
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TC (80 mg) was added in an aqueous solution (16 mL) containing 0.5% HSA. The mixture was shaken moderately at 560 oscillation per min in IKA KS 130 basic plate shaker for 6 h at room temperature, followed by lyophilization in Thermo Fischer SAVANT RVT5105 refrigerated vapour trap lyophilizer to obtain the HSA-TC as dry powder. The lyophilized product was reconstructed in 0.9% saline immediately before use. The concentration of the TC in the reconstituted injection was 5.6 mg mL−1 (characterized by inductively coupled plasma optical emission spectrometry and that of HSA in the reconstituted solution was 8.95 mg mL−1 (determined by Bio-Rad Quick Start Bradford Protein Assay kit). The size and dispersity of the HSA-TC was confirmed by DLS (Malvern Zetasizer nano Series) and electron microscopy (JEOL TEM-1400 electron microscope).
5
Sample Preparation for AIR Analysis
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AIR samples were prepared according to the literature [23 (link),26 (link)], with modifications according to a previous report [83 (link)]. Briefly, the sample was soaked in 40 mL of 80% ethanol-deionized water solution (v/v) three times (8 h, 16 h, then 8 h), with the tube sealed with a PTFE-lined screw cup and rotated on a tube rotator. After each soaking, the sample was centrifuged at 3000× g for 30 min, the supernatant was discarded, and the residue was retained for the next round of treatment. The final residue was then subjected to three rounds of washing with acetone (40 mL), followed by three rounds of washing with methanol (40 mL), with the sample being soaked in the solvent for 20 min on the tube rotator for each round. After each wash, the residue was collected by centrifugation at 3000× g for 30 min. The residue obtained after the final wash was vacuum-dried using a Savant SPD131DDA SpeedVac concentrator coupled to a Savant RVT5105 refrigerated vapor trap (Thermo Fisher Scientific Inc., MA, USA). The dry pellets were then ball-milled into a fine powder using the aforementioned ball mill system in Section 3.1.1 .
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