HUVECs were cultured in endothelial cell basal medium (EBM) supplemented with EGM-MV Single Quots (Lonza) and penicillin-streptomycin (Fisher) and maintained at 37°C in 5% CO2. Transfection with GFP-WT-MCAK and mApple-EB3 cDNAs (final concentration, 1 µg/µl) was completed using Amaxa Cell Line Nucleofector Kit V for HUVECs (Lonza), setting A-034, and experiments were performed 3–4 h later. Transfected cells (300,000–400,000 cells) were seeded in a 35-mm Petri dish (Corning) and incubated for 1.5 h to select healthy living cells from cellular debris and dead cells that were the product of the transfection procedure. Cells were treated with (−)-blebbistatin (20 µM; Cayman Chemicals) or (+)-blebbistatin (control) in dimethyl sulfoxide (DMSO; 0.001%) for 60 min before imaging and maintained in blebbistatin-containing medium plus 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) throughout imaging. Blebbistatin-containing medium and blebbistatin-treated samples were protected from light to maintain the pharmacological activity of blebbistatin and avoid phototoxicity from light-induced toxic byproducts (Kolega, 2004 (link); Sakamoto et al., 2005 (link)).
Egm mv single quots
Manufactured by Lonza
Sourced in Switzerland, United States
The EGM-MV Single Quots is a laboratory equipment product manufactured by Lonza. It is designed to provide a consistent and reliable medium for the growth and maintenance of cells in a laboratory setting. The product is intended to serve as a core function in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Nucleofector device with solution kit 5, by Lonza (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Blebbistatin, by Cayman Chemical (1 mentions)
35 mm petri dish, by Corning (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
Aurora a inhibitor 1, by Selleck Chemicals (1 mentions)
Hepes, by Lonza (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
6 protocols using egm mv single quots
1
Imaging MCAK and EB3 in HUVECs under Blebbistatin
2
Culturing Common Cell Lines for Research
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The human embryonic kidney cell line, HEK293T (#CRL-3216) and triple negative human breast cancer cell line, MDA-MB-231 (#CRM-HTB-26) were purchased from American Type Cell Culture (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) and DMEM with high glucose and sodium pyruvate from Gibco respectively, supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Gibco). Human umbilical vein endothelial cells (HUVECs) (Lonza, #C2157) from a single female donor were passaged in EBM endothelial cell basal medium supplemented with EGM-MV SingleQuots (Lonza) and grown on culture plates with bovine collagen (R&D) coating. Cells were trypsinised using 0.05% Trypsin-EDTA (Gibco) for regular passaging and grown on culture plates or flasks as an even monolayer in an incubator at 37 °C with 5% CO2.
3
Culturing Human Endothelial and Breast Cancer Cells
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Primary Human Umbilical Vein Endothelial Cells (HUVECs) were purchased (Lonza Inc.; Basel, Switzerland) and cultured in Endothelial Cell Basal Medium (EBM) medium supplemented with EGM-MV Single Quots (Lonza Inc.; Basel, Switzerland) and penicillin/streptomycin at 37 °C in 5% CO2 as previously described93 (link). Michigan Cancer Foundation-7 cells (MCF7) were purchased (ATCC; Manassas, VA) and cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 0.01 mg/mL insulin and 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37 °C in 5% CO2. Triple negative breast cancer cells (MDA-MB-231) were purchased (ATCC; Manassas, VA) and cultured in DMEM media supplemented with 10% FBS and penicillin/streptomycin at 37 °C in 5% CO2. For live imaging, HUVECs were cultured at 100,000 cells/dish on 10 µg/mL fibronectin coated 35 mm glass-bottom dishes (Cellvis, cat#: D35-20-1.5-N) and medium was supplemented with 25 mM HEPES, pH = 7.2. Transfection of cDNAs was performed using a Lonza Nucleofector Device with solution kit V (Lonza), setting A-034 for HUVECs, P-020 for MCF7 cells, and X-001 for MDA-MB-231 cells. Experiments were performed 3–4 hours post-transfection to allow time for cDNA expression.
4
Live Imaging of HUVECs on Fibronectin
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HUVECs were cultured and maintained in EBM medium supplemented with EGM-MV Single Quots (Lonza) at 37°C in 5% CO2. For live imaging, HUVECs were cultured at 50,000 cells/coverslip on 10 µg/ml fibronectin-coated glass coverslips (except for experiments using polyacrylamide substrates) and medium was supplemented with 25 µM Hepes, pH 7.2, and 30 U/ml Oxyrase. For Aurora A inhibition studies, cells were treated with 40 nM Aurora A Inhibitor I (Selleck Chemicals) for 60 min before imaging. Transfection of cDNAs and shRNA was performed using a Nucleofector Device with solution kit V (Lonza), setting A-034, and experiments were performed 6–10 h later to allow time for protein expression and/or shRNA knockdown.
5
Endothelial Cell Culture and Transfection
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HUVECs (ECs) were cultured and maintained in EBM medium supplemented with EGM-MV Single Quots (Lonza, Walkersville, MD, USA) and penicillin/streptomycin (RPI) at 37°C with 5% CO2. Transfection of siRNA and/or cDNA was performed using a Nucleofector Device with solution kit V (Lonza), setting A-034. ECs were cultured at 50,000 cells/coverslip for live-cell imaging and branching morphogenesis, or 500,000 cells/coverslip for wound-edge migration on either uncoated glass, 2D collagen-I, or 2D fibronectin-coated glass coverslips (see 2D substrate below). Experiments were performed 8-12 h after transfection to allow time for protein expression and/or siRNA knockdown. Before live-cell imaging, the exposed cell surface was supplemented with 25 µm Hepes, pH 7.2, diluted with 1× PBS (Lonza), to maintain physiological pH.
6
Endothelial Cell Culture Protocols
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HUVEC (Lonza) from a single female donor were passaged in EBM endothelial cell basal media supplemented with EGM-MV SingleQuots (Lonza). Human retinal microvascular endothelial cells (Cell System) from a male donor were cultured in Complete Classic Medium with serum and CultureBoost™ (Cell System), which were authenticated by >95% positive fluorescent staining of cytoplasmic VWF and CD31, and cytoplasmic uptake of Di-I-Ac-LDL, and <1% immunofluorescent staining of glial fibrillary acidic protein, glutamine synthetase, glial antigen 2 and platelet derived growth factor receptor-β, by the manufacturer.
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