To isolate human OECs, which are late EPCs, human umbilical cord blood (HUCB) was supplied by the Pusan National University Yangsan Hospital (PNUYH, IRB No. 05-2017-053). Total mononuclear cells (MNCs) were isolated from the HUCB by density-gradient centrifugation using Ficoll (GE Healthcare, Buckinghamshire, UK). The CD34+ cells were obtained from the MNCs using a magnetic activated cell sorting system (CD34 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The freshly isolated MNCs were then plated on cell culture dishes with 1% gelatin (Sigma-Aldrich, USA) and cultured in the EGM-2 BulletKit system (Lonza, USA). After 5 days, non-adherent cells were removed, and fresh culture medium was added. Cultures were maintained for 10–14 days until the development of cobblestone-shaped colonies. The medium was changed daily and colonies were re-plated and cultured further. For long-term priming with three small molecules, MNCs isolated from HUCB were consistently ex vivo-cultured using EGM-2 medium supplemented with 3 chemical cocktail (Fucoidan 0.1 μg/ml, TUDCA 25 μM, Oleuropein 0.5 μM), designated as OEC-3Cs. In this study, we used OECs and OEC-3Cs (passages 6–10) cultured for the same period from the same donor in all experiments.
Egm 2 bullet kit system
Manufactured by Lonza
Sourced in United States, Switzerland
The EGM-2 Bullet Kit system is a piece of lab equipment designed for cell culture applications. It is a compact and self-contained unit that provides a controlled environment for the growth and maintenance of cells. The core function of the system is to offer a standardized and consistent incubation solution for cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (2 mentions)
Ficoll, by GE Healthcare (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Penicillin streptomycin, by Welgene (1 mentions)
Fibronectin coated dishes, by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
5 protocols using egm 2 bullet kit system
1
Isolation and Priming of Human OECs
2
Isolation and Culture of Endothelial Progenitor Cells
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Total mononuclear cells (MNCs) were isolated from peripheral blood collected from healthy young human volunteers (who were recruited through relevant information) using density gradient centrifugation with Histopaque-1077 (1.077 g/mL; Sigma). Briefly, MNCs (5 × 106) were plated in 2 mL of endothelial growth medium (EGM-2 Bullet Kit System; Lonza), with supplements (hydrocortisone, R3-insulin-like growth factor 1, human endothelial growth factor, VEGF, human fibroblast growth factor, gentamicin, amphotericin B, vitamin C, and 20% fetal bovine serum) on fibronectin-coated 6-well plates. Culture medium was replaced every 4 days. Colonies of (late outgrowth) EPCs appeared between 2 and 4 weeks. The (late outgrowth) EPCs exhibited a “cobblestone” morphology and a monolayer growth pattern typical of mature endothelial cells at confluence [13 (link)].
3
Isolation and Hypoxic Culture of Human EPCs
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Human EPCs were isolated as previously described [18 (link)]. All experimental procedures have been reviewed and approved by the International Review Board (IRB) of Pusan National University Hospital (IRB No.05-2017-053). In brief, mononuclear cells (MNCs) were separated from human umbilical cord blood using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden), according to manufacturer's instructions. MNCs were cultured with the EGM-2 Bullet Kit system (Lonza, Walkersville, MD, USA) for five days in 1% gelatin-coated plates. Cultures were supplemented with 1X penicillin streptomycin (Welgene, Korea). EPCs formed spindle-shaped colonies. To generate hypoxic conditions, cells were starved with 1% FBS in EBM-2 media for 12 h, followed by a 24-h culture period at 1% O2.
4
Isolation and Culture of Endothelial Progenitor Cells
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Early EPCs were isolated as previously described [43 (link)], with some modifications. In brief, 80-mL peripheral blood samples were collected from healthy donors for the isolation of peripheral blood mononuclear cells. For rat EPCs, peripheral blood was obtained from the heart immediately before sacrifice. Ficoll-Paque™ Plus gradients were used during centrifugation to separate the fraction of mononuclear cells from other blood components, in accordance with the manufacturer’s instructions. Mononuclear cells in the low-density fraction were harvested and washed twice with PBS-ethylenediaminetetraacetic acid (2 mM). Purified mononuclear cells (1 × 106 cells/cm2) were grown on fibronectin-coated dishes (BD Biosciences) supplemented with the EGM-2 Bullet Kit system (Lonza, Switzerland), which consists of endothelial basal medium, 2% fetal bovine serum, human epidermal growth factor, human fibroblast growth factor-B, insulin-like growth factor-1, ascorbic acid and heparin. The cells were incubated in a 5% CO2 incubator at 37° C. The medium was changed every three days, and each cluster or colony was visually inspected daily through an inverted microscope at 40X magnification.
5
Isolation and Culture of Rat Endothelial Progenitor Cells
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Rat EPCs were isolated and cultured according to the previously described, with minor modifications [19 (link)]. Briefly, after collecting the mononuclear cells from the tibia and femoral bone marrow of 2-week-old rats, cells were diluted with phosphate-buffered saline (PBS), placed onto Ficoll-Paque Plus (GE Healthcare Bio-Sciences, Pennsylvania, USA), and centrifuged at 18°C at 400 g for 30 min. Then, collecting the Buffy coat mononuclear cells and washed twice with PBS. After that, the cells were resuspended with the EGM-2 Bullet Kit system (Lonza, Basel, Switzerland), which consists of endothelial basal medium, 10% fetal bovine serum, hEGF, hFGF-B, VEGF, IGF-1, heparin, and ascorbic acid. Cells were plated on fibronectin (Millipore, Billerica, MA, USA) coated onto tissue culture plastic. 24 hours after the initial plating, the medium was exchanged to remove the nonadherent cells and was exchanged every day for the first week. Colonies of EPCs appeared 7–10 days after the initial isolation, when the cells grew to 80% confluence and were serially passaged onto fibronectin–coated surfaces. EPCs were used at passages 4 for all in vitro and in vivo experiments.
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