The lentiviral particles were prepared by PEI co-transfection of HEK293T cells with the corresponding lentiviral plasmids and the packaging plasmids. The supernatants were harvested 48 h after transfection and centrifuged for 30 min at 3000 g at 4°C to remove cells and then passed through a 0.45-μm filter (Thermo Scientific) to remove cellular debris. The supernatant was further centrifuged for 2.5 h at 30,000 g at 4°C (Avanti J-26S XPI High-performance Centrifuge, Beckman Coulter). The concentrated lentiviral stocks were frozen at −80°C for future use. HEK-293T and Raji cells were infected with lentivirus and sorted by FACS.
Avanti j 26s xpi high performance centrifuge
Manufactured by Beckman Coulter
Sourced in United Kingdom
The Avanti J-26S XPI High-Performance Centrifuge is a versatile laboratory equipment used for the separation of particles, cells, and other materials based on their density and size. It features a high-speed motor and a range of rotor options to accommodate various sample sizes and applications.
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4 protocols using avanti j 26s xpi high performance centrifuge
1
Lentiviral Particle Production and Cell Transduction
2
Isolation of Small Extracellular Vesicles from Primary Human Osteoblasts
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Primary human osteoblasts isolated from three independent donors were cultured at passage 2 for a total period of 21 days. Cell culture medium incorporated FBS that has been ultracentrifuged at 120,000 × g for a period of 18 h to deplete any naturally occurring vesicles that may compromise the quality of downstream analyses (Shelke et al., 2014 (link)). sEVs were isolated from primary human every 2–3 days and pooled into three respective groups: week 1, 2, and 3. Cells and cellular debris was removed by centrifuging each sample at 2000 × g for 30 min. Five millilitre of the supernatant was carefully transferred to a sterile polycarbonate ultracentrifuge tubes (Beckman Coulter, UK) containing 2.5 mL commercial total exosome isolation reagent (ThermoFisher, UK). The solution was mixed thoroughly and incubated at 4°C to allow for sEV precipitation. Following incubation, the samples were transferred to an Avanti J-26S XPI high performance centrifuge (Beckman Coulter, UK) and spun at 10,000 × g for 60 min at 4°C in line with the manufacturer's guidelines. The supernatant was discarded, and each pellet resuspended in 300 μL filtered PBS. Fifty microliter were stored at −80°C before analysis.
3
Extraction and Profiling of Water-Soluble Peptides from Dairy Products
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The water-soluble peptide extracts (WSPE) from yogurts and heat-treated reconstituted skim milk (pHadjusted to 4.5 ± 0.05) were prepared according to Sah et al. (2014) (link) with a few modifications. Briefly, samples were centrifuged at 22,680 × g using JLA-16.250 rotor in Avanti J-26S XPI High-Performance Centrifuge (Beckman Coulter Inc., Brea, CA) for 30 min at 4°C. The supernatant was filtered using a sintered glass crucible to remove coagulated proteins, debris, and cells. The filtrate was freeze-dried using an Alpha 1-4 LSC Christ freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH) at 0.1 mbar for 24 to 36 h (main drying) and 0.08 mbar for 12 h (final drying) and stored at -80°C until further analysis. The protein content (mg/mL) of the WSPE was estimated according to Bradford (1976) using BSA (0.1-1.4 mg/mL) as standards.
The WSPE were also profiled using a reversed-phase HPLC system as described by Sah et al. (2014) (link). Briefly, the samples were loaded using a 20-μL injection loop to a Varian HPLC system (Varian Inc., Palo Alto, CA) equipped with a C-18 monomeric column (5 μm, 300 Å, 250 × 4.6 mm; Grace Vydac, Hesperia, CA) and detected eluted peptides at 215 nm.
The WSPE were also profiled using a reversed-phase HPLC system as described by Sah et al. (2014) (link). Briefly, the samples were loaded using a 20-μL injection loop to a Varian HPLC system (Varian Inc., Palo Alto, CA) equipped with a C-18 monomeric column (5 μm, 300 Å, 250 × 4.6 mm; Grace Vydac, Hesperia, CA) and detected eluted peptides at 215 nm.
4
Yogurt Peptide Extraction and Quantification
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Water-soluble peptide extracts (WSPE) were prepared by high-speed centrifugation of yogurt samples as described by Sah et al. (2014) (link). Briefly, samples were centrifuged at 22,680 × g using a JLA-16.250 rotor in an Avanti J-26S XPI High-Performance Centrifuge (Beckman Coulter Inc., Brea, CA) at 4°C for 30 min. The supernatant was collected and freeze-dried using an Alpha 1-4 LSC Christ freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) and stored at -80°C until further analysis. The protein content (mg/mL) of the WSPE was estimated according to Bradford (1976) using BSA (0.1-1.4 mg/mL) as standard.
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