Overnight cultures were subcultured into 200 ml LB and induced as above. Cultures were then pelleted via centrifugation (Beckman Coulter Avanti J‐26 XPI centrifuge; 9600g, 10 min, 4°C) before resuspension in 10 ml of sonication buffer (100 mM NaCO3 pH 7.0) and disrupted by sonication (Branson B15). Cellular debris was then collected and removed via centrifugation (Thermo Scientific Labofuge 400 R centrifuge; 3500g, 10 min, 4°C) prior to whole membrane (WM) collection by ultracentrifugation (Beckman Coulter Optima L‐100 XP ultracentrifuge; 250,000g, 45 min, 4°C). WM were then resuspended in 5 ml of MQ at 4°C.
Labofuge 400r
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Labofuge 400R is a centrifuge designed for general laboratory applications. It features a cooling system to maintain constant temperature during operation. The Labofuge 400R can accommodate a variety of sample tube sizes and has adjustable rotor options to suit different laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Avanti j 26 xpi centrifuge, by Beckman Coulter (1 mentions)
Optima l 100 xp, by Beckman Coulter (1 mentions)
Dou set elisa kit, by R&D Systems (1 mentions)
Infinite m200, by Tecan (1 mentions)
Optima xpn 80, by Beckman Coulter (1 mentions)
Edta monovette tubes, by Sarstedt (1 mentions)
Penicillin streptomycin amphotericin b, by Sangon (1 mentions)
Edta monovette, by Sarstedt (1 mentions)
Screw cap micro tube, by Sarstedt (1 mentions)
Pentra 400, by Horiba (1 mentions)
Monovettes, by Sarstedt (1 mentions)
Enzyme linked immunosorbent assay, by Mercodia (1 mentions)
13 protocols using labofuge 400r
1
Membrane Protein Isolation by Sonication
2
Isolation and Culture of Rabbit Mesenchymal Stem Cells
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The rMSCs were obtained from a male neonatal New Zealand white rabbit (0.75 kg, 1 month old). Rabbits were allowed free access to food and water at 25°C and 50–60% humidity with a 12 h light/dark cycle. The rabbit was purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The present study was carried out in strict accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of Animal Care. Bone marrow (32 ml) was aspirated from the iliac crest of the rabbit following sacrifice. The bone marrow was flushed with low glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a 1-ml syringe. The bone marrow-PBS mixture (8 ml) was centrifuged for 5 min at 1,200 × g and 20°C (Labofuge 400R; Thermo Fisher Scientific, Inc.) and the supernatant was removed. Pellets were washed with PBS (8 ml) and centrifuged at 1,200 × g again. The resultant cells were plated in a culture dish, and incubated at 37°C with 5% CO2 for 4 days. The animal protocol was approved by the Inner Mongolia Medical University Experimental Animal Management Committee (Inner Mongolia, China).
3
Extraction and Analysis of Herbal Compounds
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Based on a previous study, the LD50 of aqueous extracts of V. nigrum L. and RPA after intragastric administration were 2.566 g/kg and 160 g/kg, respectively [11 ]. In this study, the dose of V. nigrum L. was fixed at 2.566 g/kg, and the RPA dose varied from 0.2566 to 25.66 g/kg.
The doses of V. nigrum L. and RPA in each of 12 groups are shown inTable 1 . Each group was extracted using deionized water (700 mL) for 1 h during microboiling under reflux. The extracts were filtered through three layers of gauze, and the drug extraction was then repeated. The filtrates were combined and were then concentrated to 100 mL at 60−70°C under reduced pressure. Samples were then shaken, calibrated, and stored at 4°C.
All decoctions were centrifuged at 13,000 rpm for 10 min using a Heraeus Labofuge 400R refrigerated centrifuge (Thermo Scientific, USA). The supernatants were then filtered through a 0.22 μM aqueous microporous membrane and were stored at 4°C for UPLC-TOF-MS analysis. Groups were prepared in triplicate [12 , 13 (link)].
The doses of V. nigrum L. and RPA in each of 12 groups are shown in
All decoctions were centrifuged at 13,000 rpm for 10 min using a Heraeus Labofuge 400R refrigerated centrifuge (Thermo Scientific, USA). The supernatants were then filtered through a 0.22 μM aqueous microporous membrane and were stored at 4°C for UPLC-TOF-MS analysis. Groups were prepared in triplicate [12 , 13 (link)].
4
Quantification of growth factors in PRF supernatants
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The supernatants that were collected from the various PRF-based matrices at different cultivation time points were used for the quantification of different growth factors by enzyme-linked immunosorbent assay (ELISA). All collected supernatants were simultaneously centrifuged (1500 rpm; 5 min.) using a centrifuge (Thermo fisher scientific, Heraeus® Labofuge® 400 R) to exclude possible residue that could affect the photometrical measurement. Before TGF-β1 and EGF ELISA preparation, the supernatants were diluted 1:4 with the same cell culture RPMI medium used for PRF-matrices cultivation. The protein concentrations of human VEGF, TGF-β1 and EGF were determined by the Dou Set ELISA kit (Human VEGF DY293B, R&D Systems, detection range: 2000–31.3 pg/ml), HumanDou Set ELISA kit (Human TGF-β1 DY240, R&D Systems, detection range: 2000–31.3 pg/ml) and the Duo Set DuoSet ELISA kit (human EGF DY236, R&D Systems, detection range: 3.91–250 pg/mL) according to the manufacturer´s instructions. Measurements were conducted using a microplate reader (Infinite® M200, Tecan, Grödig, Austria) set to 450 nm and subtracted at 570 nm from the 450 nm measurements.
5
Exosome Isolation from Blood Plasma
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Blood plasma exosomes were isolated using a combination of ultrafiltration and ultracentrifugation. Blood samples (18 ml) were collected in K3EDTA spray-coated vacutainers. The blood cells were pelleted by centrifugation at 1,200 g (bucket rotor, Labofuge 400R, Thermo Fisher) for 20 min at 4°C. To remove the cell debris, plasma samples were centrifuged at 17,000 g (angular rotor, centrifuge 5415R, Eppendorf) for 20 min at 4°C. To remove vesicles larger than 100 nm, the supernatant was diluted 5-fold with PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.5) and filtered through 100 nm pore-size filter (Minisart high flow, 16553-K, Sartorius). For the exosome precipitation, the filtrate was centrifuged at 100,000 g (bucket rotor, Optima XPN 80, Beckman Coulter, USA) for 90 min at 4°C, the pellet was resuspended in 10 ml PBS, and re-centrifuged under the same conditions. The isolated exosomes were resuspended in 200 μl of PBS, aliquoted, frozen in liquid nitrogen, and stored at -80°C.
6
Comprehensive Metabolic Biomarker Analysis
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Plasma TAG and glucose concentrations were determined in all samples. Plasma concentrations of TC, HDL-C, LDL-C, and tumor necrosis factor α (TNF-α) were quantified from fasted samples on day 1 and day 2. Plasma insulin and nonesterified fatty acid (NEFA) concentrations were determined on day 1 in the fasted state and on day 2 at 0, 0.5, 1, 3, 4, 4.5, 6, and 8 h. Concentrations of CRP and IL-6 were measured on day 1 in the fasted state and on day 2 at 0, 2, 5, and 8 h. Plasma SOD3 and PRDX-4 concentrations were measured on day 1 in the fasted state and on day 2 at 0, 2, 4, and 8 h.
Participants rested in a semisupine position during blood sampling. Venous blood samples were drawn into precooled 9 mL EDTA Monovette tubes (Sarstedt, Leicester, UK) and immediately centrifuged at 1165g for 10 min at 4°C (Labofuge 400R; Thermo Scientific, Langenselbold, Germany). The plasma supernatant was dispensed into Cryovials and stored at −80°C for later analysis.
Participants rested in a semisupine position during blood sampling. Venous blood samples were drawn into precooled 9 mL EDTA Monovette tubes (Sarstedt, Leicester, UK) and immediately centrifuged at 1165g for 10 min at 4°C (Labofuge 400R; Thermo Scientific, Langenselbold, Germany). The plasma supernatant was dispensed into Cryovials and stored at −80°C for later analysis.
7
Cell Culture of Adherent Cells
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The BM-PBS mixture (8 ml) was centrifuged for 5 min at 1,000 rpm (Labofuge 400R, ThermoFisher, Germany) and supernatant was removed. Pellets were washed with PBS and centrifuged again. To whole cell sediment, 8 ml culture medium was added consisting of 89% DMEM/F12 (HyClone, Utah, USA), 10% fetal calf serum (FCS, HyClone), and 1% penicillin/streptomycin/amphotericin B (Sangon Biotech, Shang hai, China).
8
Venous Blood Sampling for Analysis
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Venous blood samples were collected from an antecubital vein after participants had fasted overnight. Participants lay in a semi-supine position for 5 min before samples were taken. The blood samples were drawn into pre-cooled EDTA monovettes (Sarstedt, Leicester, United Kingdom) and spun immediately in a refrigerated (4 °C) centrifuge (Labofuge 400 R, ThermoScientific, Langenselbold, Germany) at 1500 x g for 10 min. The plasma supernatant was subsequently aliquoted and stored at −80 °C prior to batch analysis.
9
Renal Tissue Homogenization and Supernatant Storage
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The isolated renal tissues were homogenized in 1:9 (w/v) physiological saline for 30
s using an FJ-200 tissue homogenizer (Shanghai Specimen and Model Factory, China).
The homogenates were centrifuged at 850 g at 0-4°C for 10 min
(Labofuge 400R; Thermo Fisher Scientific, USA), and the supernatants were kept frozen
at -75° to -80°C until they were used in subsequent assays.
s using an FJ-200 tissue homogenizer (Shanghai Specimen and Model Factory, China).
The homogenates were centrifuged at 850 g at 0-4°C for 10 min
(Labofuge 400R; Thermo Fisher Scientific, USA), and the supernatants were kept frozen
at -75° to -80°C until they were used in subsequent assays.
10
Quantifying Volatile Compounds in Thyme Leaves
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The volatile organic compounds (VOCs) of 16 plants per treatment and replication (n = 64) were extracted according to the following procedure: 100 mg (±2%) of gently oven-dried and powdered (3 intervals of 10 s at 15,000 rpm via Tube Mill control, IKA®, Staufen, Germany) thyme leaves were transferred into 2 mL screw cap micro tubes (SARSTEDT AG & Co. KG, Nümbrecht, Germany) including two steal grinding balls (Ø 2 mm). The plant material was homogenized in 1.0 mL of isooctane (containing 1:40,000 (v/v) 6-methyl-5-penten-2-one as internal standard) for 10 min at 30 rps with a ball mill (MM400, Retsch®, Haan, Germany). After 10 min of ultra-sonication (Sonorex RK 106, BANDELIN electronic GmbH & Co. KG, Berlin, Germany) and 10 min of centrifugation at 13,000 rpm (Heraeus™ Labofuge™ 400 R, Thermo Scientific™, Osterode, Germany) at 22 °C respectively, the supernatants were transferred into GC-vials and stored at −70 °C until analysis.
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