Cells in osteogenic medium on Day 14 were lysed in 1% Triton X-100 in PBS and centrifuged at 400× g for 5 min (Heraeus Labofuge 400R, Thermo Fisher Scientific). Then, the supernatants (cell lysate solutions) and cell pellets were collected and kept at −80 °C for further analyses. Subsequently, the protein content in the cell lysate solutions was measured using a Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions. Then, the levels of ALP activity in total protein cell lysate solutions were measured using Alkaline Phosphatase Yellow Liquid Substrate for ELISA (Sigma-Aldrich) following the manufacturer’s instructions [40 (link)].
Heraeus labofuge 400r
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Heraeus Labofuge 400R is a compact, refrigerated centrifuge designed for general laboratory use. It features a brushless, maintenance-free motor and a high-capacity rotor capable of handling a variety of sample sizes. The centrifuge provides precise temperature control and can reach speeds up to 15,000 rpm, enabling efficient separation of samples.
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Lab products found in correlation
Dc protein assay kit, by Bio-Rad (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 fgf2, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Amphotericin b, by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Sorvall wx ultra series 80, by Thermo Fisher Scientific (1 mentions)
Millipore water, by Merck Group (1 mentions)
Potassium edta monovettes, by Sarstedt (1 mentions)
Dlp00, by R&D Systems (1 mentions)
Twinguard, by Panasonic (1 mentions)
S monovette system, by Sarstedt (1 mentions)
Screw cap micro tube, by Sarstedt (1 mentions)
Sonorex rk 106, by Bandelin (1 mentions)
8 protocols using heraeus labofuge 400r
1
Osteogenic Cell Lysis and ALP Assay
2
Isolation and Culture of hBMSCs
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All procedures were approved by the Institutional Ethical Committee of Hannover Medical School (Hannover, Germany). After written informed consent was obtained, bone marrow aspirates were collected from seven healthy human donors (four males and three females; mean age, 29±3.5 years) who underwent exposure of their iliac crests during routine orthopedic procedures. Isolation and cultivation of hBMSCs was performed as per our previously described protocol (21 (link)). Briefly, the cells were purified by density gradient centrifugation at 1,200 × g for 20 min at 4°C (Heraeus Labofuge 400R; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were then cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium containing L-glutamine (Biochrom GmbH, Berlin, Germany) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 5 µg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 3 ng/ml fibroblast growth factor-2 (FGF-2; PeproTech, Inc., Offenbach, Germany), 100 U/ml penicillin/streptomycin (Gibco) and 0.5 µg/ml amphotericin B (Biochrom GmbH) at 37°C and 5% CO2 in humidified atmosphere. After reaching confluence, cells were lysed with 0.05% trypsin (Gibco) and combined, and then the cell pool was subcultured. The hBMSCs from the third passage were used for the experiments.
3
Isolation of Extracellular Vesicles from Mouse Plasma
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Peripheral blood from deeply anesthetized reporter or double-transgenic mouse previously injected with LPS was collected during perfusion. Platelet-free plasma was isolated through centrifugation steps (Heraeus Labofuge 400R; Thermo Fisher Scientific, Waltham, MA, USA) of 2,500 rpm for 15 min at RT twice and diluted 1:1 with 10 mM HEPES buffer (A6916,0125; AppliChem GmbH, Darmstadt, Germany) in Millipore water (EMD Millipore, Burlington, MA, USA). The solution was 0.45-μm filtered to remove larger particles and mixed with polyethylene glycol (PEG) 8000 (Rotipuran, 0263.1, Roth) solution 1:5 for precipitating vesicles during an overnight incubation at 4°C. Samples were centrifuged at 1,500 × g for 30 min at 4°C, and pellets were resuspended in 10 mM HEPES buffer for an ultracentrifugation step (Sorvall WX Ultra Series 80; Thermo Fisher Scientific) at 100,000 × g for 90 min. The pellets were resuspended in 10 mM HEPES buffer and kept at 4°C for immediate use or at −80°C for longer storage.
4
Plasma Biomarker Quantification Protocol
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Blood samples were collected into ice-cooled potassium EDTA monovettes (Sarstedt, Leicester, UK) and were spun immediately in a refrigerated centrifuge (Heraeus Labofuge 400R, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 10 min (2383× g). Plasma was then removed and aliquoted for storage at −80 °C. To prevent protein degradation by cysteine-proteases, samples (monovettes) for acylated ghrelin were pre-treated with a 50 µL solution comprising 0.1 M phosphate buffered saline, P-hydroxymeruribenzoic acid and 10 M sodium hydroxide. After centrifugation, plasma was acidified with 1 M hydrochloric acid and re-centrifuged for five min. Commercially available enzyme-linked immunosorbent assays (ELISAs) were used to measure plasma concentrations of leptin (DLP00, R & D Systems, Oxford, UK; CVintra 6.8%), acylated ghrelin (97751 SCETI, Tokyo, Japan; CVintra 4.3%) and total PYY (EZHPPPT66K, Millipore, Watford, UK; CVintra 2.4%). All samples from each participant were analysed on the same assay plate to reduce variation.
5
Venous Plasma Glucose Dynamics
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Venous blood samples were collected via cannula at baseline (before breakfast), immediately after breakfast (t = 0 min) and every 15 min from t = 0 min until 240 min for venous plasma glucose (VPG) measurement. Venous blood was drawn into pre-chilled 4.9 mL K3 EDTA tubes using the Sarstedt S-Monovette® system (Sarstedt, Nümbrecht, Germany). These blood samples were then centrifuged at 3500 rpm (2054× g) for 10 min (Heraeus Labofuge 400R, Thermo Fisher Scientific, Waltham, MA, USA). The separated plasma was stored in a −80 °C freezer (TwinGuard, Panasonic, Kadoma, Japan) until biochemical analysis. VPG concentrations were assayed using the ABX Pentra Glucose PAP CP kit by colorimetry on a Pentra C400 clinical chemistry analyser (HORIBA Medical, Montpellier, France), with an intraassay coefficient of variation of 0.5%.
6
Volatile Compound Extraction from Dried Basil Leaves
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After samples were gently air-dried in a drying oven at 30 °C for ≤ 7 days until stable mass was attained and shortly stored under cool, dry and dark conditions, volatile compounds of basil leaves were extracted according to the following procedure: 100 mg (± 2 %) of dried and powdered (3 intervals of 10 seconds at 15,000 rpm via Tube Mill control, IKA®, Staufen, Germany) basil leaves from two basil pots (of the same cultivar, light treatment, harvest date and experimental block) were transferred into 2 mL screw cap micro tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany) including two steal grinding balls (Ø 2 mm) and homogenized in 1.0 mL of high-performance liquid chromatography grade isooctane (Th. Geyer, Renningen, Germany) [containing 1:2000 (v/v) carvacrol as internal standard] for 10 minutes at 30 rps with a ball mill (MM400, Retsch®, Haan, Germany). After 10 minutes of ultra-sonification (Sonorex RK 106, Bandelin electronic GmbH & Co. KG, Berlin, Germany) and 10 minutes 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 (N = 256 samples).
7
Feline Blood Plasma Collection
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For each cat at the time of enrollment, peripheral blood was collected by jugular or cephalic venipuncture, with 4–6 mL placed in serum tubes containing a clot accelerator and granule serum separator (Aquisel, Barcelona, Spain). These samples were centrifuged at 790× g for 10 min (Heraeus Labofuge 400R, Thermo Fisher Scientific, Waltham, MA, USA) to obtain the serum that was stored at −80 °C until used.
8
Natural Product Library Screening
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A total of 297 compounds were screened and were derived from three different natural product libraries which are named after the groups that provided them KOM (Microbial Communication, HZI, Germany), MWIS (Microbial Drugs, HZI, Germany) and SAAR (Helmholtz-Institute of Pharmaceutical Research, Saarbrücken, Germany).
In case of KOM and MWIS, the stocks of the compounds were provided in methanol. These libraries were screened at a final concentration of 5 μg and 0.5 μg per ml in triplicates. Compounds from SAAR were provided on a polypropylene micro-titre plate with 5 μl of the compound at 1 mM concentration in DMSO. Plates were stored at − 80 °C. Shortly before screening, the plate was retrieved from − 80 °C and thawed at room temperature. To pool the compounds to the bottom, the plate was centrifuged in a microplate centrifuge (Thermo scientific Heraeus labofuge 400 R) at 1500 rpm for 5 min and the compounds were transferred to an optical bottom polystyrene black microtitre plate for testing. The final concentration of the compounds from the SAAR library was 25 μM.
In case of KOM and MWIS, the stocks of the compounds were provided in methanol. These libraries were screened at a final concentration of 5 μg and 0.5 μg per ml in triplicates. Compounds from SAAR were provided on a polypropylene micro-titre plate with 5 μl of the compound at 1 mM concentration in DMSO. Plates were stored at − 80 °C. Shortly before screening, the plate was retrieved from − 80 °C and thawed at room temperature. To pool the compounds to the bottom, the plate was centrifuged in a microplate centrifuge (Thermo scientific Heraeus labofuge 400 R) at 1500 rpm for 5 min and the compounds were transferred to an optical bottom polystyrene black microtitre plate for testing. The final concentration of the compounds from the SAAR library was 25 μM.
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