Peripheral blood mononuclear cells were isolated from the peripheral blood of patients with CAD and healthy donors using ficoll density gradient centrifugation. The cells were then cultured on fibronectin-coated six-well plates in endothelial basal medium (Cambrex, Walkersville, MD, USA) supplemented with vascular epidermal growth factor (Preprotech, Rocky Hill, NJ, USA), human recombinant long insulin-like growth factor-1, ascorbic acid, cortisol and 20% FBS (Hyclone, South Logan, UT, USA) at 37 °C in a 5% CO2 incubator. After 4 days, non-adherent cells were removed by washing with PBS. Adherent cells (attached early EPCs) were incubated in fresh medium every 3 days and were used for the subsequent experiments. These cells had elongated spindle-shape morphology and their phenotype was confirmed by assessing the surface markers with flow cytometry analysis (Becton Dickinson, Franklin Lakes, NJ, USA). FITC-conjugated antibodies of CD31, CD34 and CD45 (Abcam, Cambridge, MA, USA) were used [62 (link)].
Endothelial basal medium
Manufactured by Cambrex
Sourced in United States
Endothelial basal medium is a cell culture medium designed to support the growth and maintenance of endothelial cells. It provides the essential nutrients and growth factors required for the in vitro cultivation of endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Vascular epidermal growth factor, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Flow cytometry analysis, by BD (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Biocoll, by Harvard Bioscience (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
6 protocols using endothelial basal medium
1
Isolation and Characterization of Early Endothelial Progenitor Cells
2
Isolation and Characterization of Endothelial Progenitor Cells
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First, 2 ml peripheral blood was collected from each patient with CAD (n = 20) and healthy donors (n = 20). Through ficoll density gradient centrifugation, the peripheral blood mononuclear cells were isolated. These cells were then cultured on six-well plates coated with fibronectin for 24 h before transplanting, and in the endothelial basal medium (Cambrex, Walkersville, MD, USA) supplemented with human recombinant long insulin-like growth factor-1, ascorbic acid, vascular epidermal growth factor (Preprotech, Rocky Hill, NJ, USA), cortisol and 20% FBS (Hyclone, South Logan, UT, USA) at 37 °C in a 5% CO2 incubator. To screen the EPCs, non-adherent cells were removed by washing with PBS after 4 d, and the adherent cells (attached early EPCs) were incubated in fresh medium every 3 d. The morphological characteristic of EPCs was elongated spindle-shape. To further identify their phenotype, flow cytometry analysis (Becton Dickinson, Franklin Lakes, NJ, USA) was conducted to assess the surface markers. FITC-conjugated antibodies of CD31, CD34 and CD45 (Abcam, Cambridge, MA, USA) were used (Ikutomi et al., 2015 (link)).
3
Isolation and Culture of Mononuclear Cells
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Mononuclear cells were isolated by density gradient centrifugation with Biocoll (Biochrom, Berlin, Germany) from 20 mL of venous blood as previously described in detail (4 (link), 28 (link)). Briefly, after isolation, 4×106 mononuclear cells were placed on 24-well culture dishes blankets with human fibronectin (Sigma-Aldrich, Munich, Germany) and kept in endothelial basal medium (Cambrex, Walkerville, MD, USA) enriched with endothelial growth medium and 20% fetal calf serum. After the 4th day in culture, non-engrafted cells were detached by thorough washing with phosphate-buffered saline (PBS).
4
Histamine-Induced Endothelial Barrier Regulation
5
HUVEC Cell Culture Protocol
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Human umbilical vein endothelial cells (HUVECs) were purchased from Cascade Biologics (Portland, OR) and grown in endothelial basal medium (Clonetics Inc Walkersville, MD) supplemented with 2% foetal calf serum (FCS) and growth factors, penicillin (100 u/mL), and streptomycin (100 µg/mL). All cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Cells were grown to 70%‐80% confluent with starvation before being treated with different agents.
6
Culturing Human Umbilical Vein Endothelial Cells
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As described previously,18 human umbilical vein endothelial cells (HUVECs) were purchased from Cascade Biologics (Portland, OR) and grown in endothelial basal medium (Clonetics Inc Walkersville, MD). In all experiments, cells were between passages 3 and 8. All cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
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