With agreement from the ethics committee of Jinan Central hospital Affiliated to Shandong University, the principal investigator designed the experiment and received ethical approval. Written informed consent was obtained from each donor. Human peripheral blood samples (50–100 mL/unit) were collected from 26- to 42-year-old (averaged 31 ± 6) healthy donors (three male and two female) at Jinan Central Hospital Affiliated to Shandong University. Mononuclear cells were isolated with Ficoll-Hypapue (γ = 1.077, Sigma), and red blood cells were removed using red blood cell lysis buffer (eBioscience). The remaining mononuclear cells were washed three times with PBS and seeded in 150 × 15 or 60 × 15 mm Petri dishes (BD Falcon™) at 1–2.5 × 106 cells/mL. Cells were cultured in serum-free culture medium (Lonza, Allendale, NJ) and incubated at 37°C with 8% CO2 in air (Zhao et al., 2007 (link); Li et al., 2012 (link)).
Serum free culture medium
Manufactured by Lonza
Sourced in Italy
Serum-free culture medium is a laboratory product designed to support the growth and maintenance of cells in vitro. It provides the necessary nutrients and growth factors for cell cultivation without the use of serum, which can introduce variability and potential contamination. The core function of this medium is to create a controlled and defined environment for cell culture.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using serum free culture medium
1
Isolation and Culture of Mononuclear Cells
2
Isolation and Characterization of Multicellular Spheroid-Derived Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Conditioned medium, MVs and SN were obtained by multicellular spheroids only. Multicellular spheroids were cultured for 3 days with 1 mL per well of a serum-free culture medium (Lonza, Milano, Italy) added with 1% penicillin (100 IU/mL)-streptomycin (100 mg/mL) and 2 mM l-glutamine. Conditioned media of three different samples were collected, pooled and centrifuged at 2500 g for 20 min to remove the cellular debris. A portion of this CM in toto was stored at -20°C. To obtain MVs and SN, another aliquot of CM was ultracentrifuged (Beckman Coulter Optima L -100 K) at 100,000 g for 1 h at 4°C and SN was collected and stored at -20°C for later use. The pellet was washed in serum-free medium 199 containing N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES) 25 mM and submitted to a second ultracentrifugation under the same conditions. The resulting pellet, composed of MVs, was fractionated for MVs analysis or used for the in vitro test.
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