The 4T1 cells were grown in RPMI-1640 supplemented with 10% of ultracentrifuged FBS and maintained at 37 °C and 5% CO2 in a humidified atmosphere. When the 4T1 cells reached an approximate confluence of 80%, the supernatant was removed from the cell culture flask T-75. The exosome isolation reagent was added to the supernatant (1:2 v/v ratio, respectively) and kept in a refrigerator for 15 h. After that time, the mixture was centrifuged at 10,000× g for 1 h at 4 °C using a centrifuge from Thermo Scientific, model Heraeus Multifuge X 1R. The pellet was dissolved in a mixture of chloroform and methanol (1:1 v/v ratio).
Heraeus multifuge x1r
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Heraeus Multifuge X1R is a high-performance, general-purpose centrifuge designed for a wide range of laboratory applications. It features a robust, durable construction and offers a maximum speed of 15,000 rpm and a maximum relative centrifugal force (RCF) of 21,382 x g. The Multifuge X1R is equipped with a user-friendly control panel and provides a variety of safety features to ensure reliable and consistent operation.
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Lab products found in correlation
Lindberg blue m, by Thermo Fisher Scientific (3 mentions)
Complete dmem, by Thermo Fisher Scientific (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Penicillin, by Solarbio (2 mentions)
Streptomycin, by Solarbio (2 mentions)
Polygard cr opticap xl capsule, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Aam blg 1, by RayBiotech (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Beh c18, by Waters Corporation (1 mentions)
1260 hplc system, by Agilent Technologies (1 mentions)
Milli q direct 8, by Merck Group (1 mentions)
Human vegf duoset elisa, by R&D Systems (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ev depleted fbs, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
26 protocols using heraeus multifuge x1r
1
Exosome Isolation from 4T1 Cell Culture
2
Bacterial Biomass Preparation and Preservation
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Bacterial biomass was separated from the growth medium by centrifugation (Heraeus Multifuge X1R, Thermo Scientific, Waltham, MA, USA) at 2490g 4°C for 10 min and washed with distilled water three times. Further, at the last washing step, 100–500 μL of distilled water was added to the cell pellet and re‐suspended. About 10 μL of the homogenized bacterial suspension was pipetted onto the IR‐light‐transparent silicon 384‐well silica microplates (Bruker Optics GmbH, Ettlingen, Germany) in three technical replicates, and dried at room temperature for at least 2 h before the analysis (Smirnova et al., 2021 (link); Smirnova et al., 2022 (link)). The remaining bacterial biomass was freeze‐dried (Labconco, USA) for 72 h until constant weight, and stored at −20°C. Freeze‐dried biomass was used for lipids extraction.
3
Establishing cell lines for LAG-3 study
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CHO-K1 and Jurkat leukemia T cells overexpressing hLAG-3 were established by lentiviral transfection and selected with 1 mg/mL puromycin. They were cultured in RPMI-1640 medium (Gibco, Grand Island, USA) containing 10% heat-inactivated FBS (BI, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, China) in 37 °C and 5% CO2 incubator. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were diluted with PBS (pH 7.2) and separated with Ficoll density gradient and centrifuge (2000 rpm, 30 min, 25 °C) (Thermo Fisher Scientific, Heraeus Multifuge X1R, USA)45 (link). The isolated cells were resuspended at 1 × 106 cells/mL in complete RPMI-1640 medium. To isolate the mouse spleen lymphocytes, the mouse spleen was extracted and digested into single cells by collagenase IV (17104-019, Gibco) and DNase I (Sigma, USA). We adjusted the cells density into 2 × 106 cells/mL and cultured in complete RPMI-1640 medium. The HepG2 cells and MC38 cells were cultured in complete DMEM (Gibco, Grand Island, USA) medium.
The antibodies used for flow cytometry (BD FACS Celesta, USA) are listed in Supporting InformationTable S1 . And proteins used for MST and blocking assay are listed in Supporting Information Table S2 .
The antibodies used for flow cytometry (BD FACS Celesta, USA) are listed in Supporting Information
4
Plasma Proteome Analysis of Irradiated Mice
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Plasma was extracted from the whole blood of normal controls and mice irradiated with 0.05 Gy (×10), 0.1 Gy (×5), and 0.5 Gy (×1) via centrifugation at 1,000×g for 15 min (Heraeus Multifuge X1 R; Thermo Fisher Scientific, Waltham, MA, USA). Twenty microliters of plasma were used for the biotin label-based L-series mouse antibody array (AAM-BLG-1; Raybiotech, Atlanta, GA, USA), which was performed according to the manufacturer’s instructions.
5
Synthesis of TiO2-FeS2 Nanocomposites
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Fe((NO3))3·9H2O (8 mmol), thioacetamide (100 mmol), and TiO2 (8 mmol, P25) were dissolved in 10 mL DMF and then transferred to a Teflon container. The Teflon container was fixed in an autoclave reactor and placed in a hot-air oven (JOV-40) at 180°C for 18 h. After cooling to room temperature, the solution was centrifuged (Heraeus multifuge X1R, Thermo Scientific) at 4000 rpm for 10 min and washed with ethanol to remove excess organic residues. Afterward, the primary product of FeS2-NCs was collected and dried in the oven. A mixture of the primary product of FeS2-NCs (0.1 g) and sulfur powder (0.3 g) was placed in a furnace (Thermofisher Lindberg Blue M) at 500°C for 1 h to obtain the final product of TiO2-FeS2 NCs. For antibacterial tests, TiO2 NPs (0.4 mg), FeS2 NPs (0.4 mg), and TiO2-FeS2 NCs (0.8 mg) were, respectively, drop-cast onto CFP (CeTech) with dimensions of 1 x 2 cm.
6
Cellulase Adsorption on Delignified Substrates
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The experiments of productive and non-productive adsorption of cellulase on DA-SCB were conducted in 0.05 mol/L citric acid/sodium citrate buffer of pH 4.8 at 50 °C, using DA cellulose and DA lignin, respectively, as substrates. The substrate loading was 2% (w/v) and the enzyme loading was 4.30 mg protein/g substrate (7.5 FPU/g substrate) [20 (link), 44 (link)]. Firstly, the substrates and buffer were pre-equilibrated for 2 h. Secondly cellulase was added to the solution containing substrate and buffer. After adsorption for 2 h, the mixture was centrifuged (Heraeus Multifuge X1R, ThermoFisher Scientific, America) at 7441g (8000 rpm) for 15 min, and then the protein amount in supernatant was measured. The productive or non-productive adsorption of cellulase was calculated by subtracting the amount of free protein in the supernatant from the total cellulase amount added to the cellulase–cellulose or cellulase–lignin adsorption system (4.30 mg protein/g substrate). All experiments and assays were performed in triplicate.
7
Plasma Profiling of Radiation-Induced Mortality
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Whole blood from the tail vein was sampled into EDTA-containing tubes 24 hours before and 24 hours after IR. Plasma was extracted from whole blood through centrifugation at 1000 × g for 15 minutes (Heraeus Multifuge X1R; Thermo Scientific, Rockford, Illinois). Six mice that survived and 6 mice that died on day 13 were selected, and plasma samples were pooled for 2 animals (ie, 3 plasma samples were obtained from 6 mice that survived, and 3 plasma samples were obtained from 6 mice that died). Twenty microliters of plasma were used for the biotin label-based L-series mouse antibody array (AAM-BLG-1; Raybiotech, Atlanta, Georgia), according to the manufacturer’s instructions.
8
Culturing and Seeding MDA-MB-231 Cells for Microdevice Experiments
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MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC) and were grown according to the instructions provided by the ATCC. In details, the cells were cultured in RPMI 1640 (11875–093, Invitrogen) with 10% FBS (Corning-Cellgro) and 1.0% penicillin (1.0×104 units/ml) and streptomycin (10 mg/ml). Cells were seeded and grown in an incubator at 37.0°C, 5.0% CO2 and 80.0% humidity. Before loading into the microchips, MDA-MB-231 cells were grown in flasks in a monolayer to 80% confluence and then harvested with 0.25% trypsin-EDTA (Mediatech). These cell suspensions were then centrifuged (Heraeus multifuge X1R, Thermo, GER) at 400 x g for 5 minutes. The centrifuged cells were pelleted and re-suspended in RPMI 1640 before being loaded into the microdevice. Each cell chamber was loaded with approximately 104 cells.
9
Synthesis and Purification of Internal Standard
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For the preparation of the internal standard (ISTD) (1,2-bis-naphthoylethanediol), 0.8 g of 2-NCl and 2.4 g of DMAP were dissolved in 4.5 mL of methylene chloride in a 40-mL glass centrifuge tube equipped with a screw cap in an ultrasonic bath for 2 min. Five grams of ethane-1,2-diol were added, and the tube was briefly vortexed and stored for 1 week at 50 °C in a drying oven. After cooling to room temperature, 5 mL of n-hexane were added, and the tube was briefly vortexed. Excess of derivatization reagent was removed by twofold shaking with 7 mL of 2.5 M hydrochloric acid and twofold shaking with 7 mL of saturated NaHCO3 solution for 10 min on a small shaking device (VXR basic, IKA) at 2200 min−1. After each shaking step, centrifugation followed for 2 min at 3000 rpm and 18 °C (Heraeus Multifuge X1R, Thermo Scientific, Dreieich, Germany). The organic phase was transferred into a 12-mL screw-capped glass vial and the solvent was completely removed under a stream of nitrogen. The viscous residue was finally dissolved in 500 μL of TBME. The ISTD working solution was stored at room temperature.
10
Quantitative LC-MS/MS Analysis Protocol
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The LC-MS/MS system consisted of an Agilent 1260 HPLC system equipped with a cooled autosampler (4°C) and an Agilent 6460 electrospray ionization-triple quadrupole mass spectrometer (Agilent Technologies, USA). The chromatographic column was BEH C18 (Waters, 2.1 × 50 mm, 1.7 μm) (Waters Corporation, USA). Ultrapure water was prepared using the Milli-Q Direct 8 (E. Merck, Darmstadt, Germany) water purification system. A Heraeus Multifuge X1R (Thermo Fisher Scientific, USA) high-speed refrigerated centrifuge was used for the centrifugation. SHIMADZU-AUW120D (Shimadzu Corporation, Japan) was used for the weighting.
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