CB and PB ECFC-derived cells (1 × 105 cells) were plated onto 6 well plates in complete EGM-2 media (Lonza Biologics) overnight. The following day, media were removed and cells transfected with 10 nM mimic non-targeting control, and the relevant miRNA mimic or 10 and 50 nM inhibitor non-targeting control and the relevant miRNA inhibitor (Dharmacon, Lafayette, CO, USA) using Oligofectamine transfection reagent according the manufacturer’s protocol (Life Technologies). After 5 hr transfection, the transfection reagent was replaced with complete EGM-2 media (Lonza Biologics). Cells were harvested for other experiments at 48 hr after transfection.
Complete egm 2 media
Manufactured by Lonza
Complete EGM-2 media is a cell culture medium designed for the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors required for the optimal in vitro culture of endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Oligofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Cat no cap 0006gfp, by Angio-Proteomie (1 mentions)
Quick coating solution, by Angio-Proteomie (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Egm 2mv medium, by Lonza (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
5 protocols using complete egm 2 media
1
miRNA Transfection of ECFC Cells
2
Cell Culture Protocols for Cancer Research
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Triple negative human breast adenocarcinoma cells (MDA-MB-231), the human fibroblast cell line and the human embryonic kidney cell line HEK-293 were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum, 1% penicillin–streptomycin and 0.1% amphotericin B. Murine triple negative breast cancer cell line 4T1 cells were cultured in RPMI-1640 with 10% bovine serum and 1% penicillin–streptomycin. Primary cells HUVEC were cultured using complete EGM2 media (Lonza) and were seeded in plates coated with 0.1% gelatin.
3
Culture and Expansion of Human Aortic Endothelial Cells
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Human aortic endothelial cells (hAEC) were chosen as an EC model for their relevance in cardiovascular function and disease51 (link),52 (link). Enhanced green fluorescent protein (eGFP) transfected hAEC were purchased from Angio-Proteomie (Boston, MA) (CAT No. cAP‐0006GFP, LOT No. 201209701SR). Cells were cultured according to manufacturer protocol. Briefly, eGFP-hAEC were thawed, suspended in complete EGM-2 media (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) (EGM-2F) and plated in a T-75 flask pre-coated with quick coating solution (Angio-Proteomie). All cells were cultured at 37 °C and 5% CO2, and upon reaching 80% confluency, were passaged at a 1:4 ratio. All experiments utilized cells from passage 4–6 (P4-P6).
Human AECs were purchased from Lonza (Walkersville, MD), (CAT No. CC-2535, LOT No 0000303583) and cultured in the same way as described for eGFP-hAEC.
Human AECs were purchased from Lonza (Walkersville, MD), (CAT No. CC-2535, LOT No 0000303583) and cultured in the same way as described for eGFP-hAEC.
4
Endothelial Cell Culture Protocols
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hCMEC/D3 (human cerebral microvascular endothelial cell line) cells used from passages 30 to 37 after they were grown to confluence on collagen‐coated plates as previously described.29 Cells were cultured in EGM‐2 MV medium, with all supplements added as directed by the manufacturer (Lonza). HUVEC (Human Umbilical Vein Cells, Lonza), used from passages 4 to 7, were grown to confluence on fibronectin‐coated plates, and maintained using complete EGM‐2 media (Lonza). HKi2, 10 mM in DMSO, was maintained at room temperature for 30 min rotating before use. Cells were then treated with HKi2 at the concentrations and times indicated for each experiment. Treatments with 50 or 75 μM HKi2 were doses selected for maximal effects on hCMEC/D3 and HUVECs, respectively (Figures 6 and 7 ). Vehicle control cells were treated with the same concentration of DMSO. Cells were maintained at 37°C in 5% CO2. Chinese hamster ovary (CHO) cells were grown on glass coverslips and maintained in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L‐glutamine. CHO cells were co‐transfected with EGFP‐KRIT1 FERM domain, and either mito‐mCherry‐HEG1 or HEG1 ΔYF cytoplasmic tail expression plasmids. Mito‐mCherry‐HEG1 wild‐type or ΔYF were cloned into pcDNA3.1(‐) as previously reported.28 Human KRIT1 cDNA encoding EGFP‐tagged KRIT1 FERM domain was previously described.4
5
Wound Healing Assay Protocol
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Cells were grown to full confluence in 24-well plates in complete EGM-2 media (Lonza) and were incubated overnight. Cell cultures were scratched with a 10 µL sterile pipette tip and extensively washed with PBS to remove detached cells and debris. One scratch per well was generated, and then center-imaged at 5× magnification, using a Zeiss Axio Observer 200 M microscope equipped with a Zeiss AxioCam MRm camera with maximum contrast (Carl Zeiss AG, Feldbach, Switzerland). Cells were then incubated in serum-reduced or full-growth medium. Images were taken at 0, 2, 4, 8, 24, and 48 h after the scratch. The wound areas were calculated and analyzed with Image J, version 1.8.0 (NIH, Bethesda, MD, USA) and expressed as a percentage of the day 0 value.
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