EA.hy926 cells were cultured in DMEM (4.5 g l−1 glucose) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and glutamine (2.8 mM) and HAT-supplement (hypoxanthine, aminopterin, thymidine). EA.hy926 with lentiviral shRNA knockdown of LDLR or control shRNA (scramble) were cultured in regular EA.hy926 media with 500 ng ml−1 puromycin. HUVEC were cultured in M199 supplemented with endothelial cell growth supplement (Yale VBT Core Facility), 10% FBS, penicillin/streptomycin (1:100) and glutamine (2.8 mM) and used up to passage 4. For siRNA transfections HUVEC were cultured in EGM-2 media (Lonza) with 5% FBS after seeding in 384-well plates as it improves the transfection dramatically. MLEC were cultured in EGM-2 media (Lonza) with 20% FBS. HCAEC were cultured in EGM-2MV media and used in passages 5–8. HeLa cells and Ldlr-KO MEF were kept in DMEM (4.5 g l−1 glucose) supplemented with 10% FBS and penicillin/streptomycin.
Egm 2 media
Manufactured by Lonza
Sourced in United States, Switzerland, United Kingdom
EGM-2 media is a cell culture medium formulated for the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors to support the in vitro culture of endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Huvecs, by Lonza (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Huvecs, by Thermo Fisher Scientific (4 mentions)
Vegf165, by Thermo Fisher Scientific (3 mentions)
Egm 2, by Lonza (3 mentions)
Dynabead, by Thermo Fisher Scientific (3 mentions)
Matrigel, by Lonza (3 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (3 mentions)
Huvecs, by Corning (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Matrigel, by BD (3 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (3 mentions)
Hmvec, by Lonza (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Tryple express, by Miltenyi Biotec (2 mentions)
Chir99021, by Bio-Techne (2 mentions)
141 protocols using egm 2 media
1
Cell Culture Protocols Across Cell Types
2
Cloning and Transfection of VEAL2
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The plasmid pcDNA 3.1+ was used to clone full‐length VEAL2 or veal2 using EcoRI and NotI restriction sites. The EcoRI and NotI restriction enzyme sites were introduced at the ends of full‐length VEAL2 or veal2 in vitro using specific primers (primer details given in Appendix Table S3 ). Then, this fragment was cloned into a linearized pcDNA 3.1+ vector. Finally, the sequence of the identified recombinant plasmid was confirmed by Sanger sequencing. The transfection was performed using HUVEC nucleofector kit (vpb‐1002) (Lonza, USA) according to the manufacturer's protocol as follows: (i) HUVECs were cultured in EGM2 media (Lonza, USA) (P2‐P5) in T‐75 flasks at 37°C and with 5% CO2. (ii) 1 M cells were pelleted down and dissolved in 100 μl electrolyte in a cuvette and nucleofected using A‐034 program in nucleofector machine from Lonza, USA. (iii) After nucleofection, cells were dissolved in additional 900 μl HUVEC EGM‐2 media and aspirated to a fresh tube. These transfected cells were further used for various assays.
3
Maintenance of Pluripotent Stem Cells
4
Maintenance of Pluripotent Stem Cells
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Human ESCs, H9 (WiCell) and human iPSCs, BJFF (provided by Prof. Sanjay Jain at Washington University) are maintained in feeder-free culture using StemFit ® Basic02 (Ajinomoto Co., Inc.) supplemented with 10 ng/ml FGF2 (Peprotech) as previously reported1 (link). Human glomerular microvascular endothelial cells (GMECs), RFP expressing (Angio-Proteomie) are cultured using EGM2 media (Lonza) and used up to passage 9. Human umbilical vein endothelial cells (HUVECs), RFP expressing (Angio-Proteomie) are cultured using EGM-2 media (Lonza) and used up to passage 9. Human neonatal dermal fibroblasts (HNDF), GFP expressing (Angio-Proteomie) are cultured per supplier’s instructions and used up to passage 15.
5
Immortalized Adipose Stem Cell Printing
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A hTERT-immortalized human adipose tissue–derived
stem cell
line (ASC/TERT1) (Evercyte) was transfected with green fluorescent
protein (GFP) to obtain permanently transfected green-labeled cells
following a previously established protocol.22 (link) ASCs-GFP were cultured in endothelial cell growth (EGM-2) media
(Lonza) supplemented with 10% fetal bovine serum (Sigma). The cells
were maintained in an incubator (high humidity, 5% CO2,
37 °C), and the medium was changed every second day. To enable
printing in the presence of living cells, the cells were trypsinized
with 0.5% trypsin–EDTA and resuspended at 1 x 106 cells/mL in 7.5 and 10 w/v % RCPhC1-NB/SH solution supplemented
with 2 mol % DAS with respect to the photo-cross-linkable functionalities.
The solution was pipetted into a silicone mold in the glass bottom
methacrylated Ibidi dishes. Cubes of 200 μm × 200 μm
× 200 μm were printed using a writing speed of 1000 mm/s
and laser powers of 50, 70, and 100 mW. The proliferation rate was
determined by counting the cells in the cubes after days 1, 3, and
6, and the data were normalized to day 1. To this end, ImageJ software
was used to assess maximum intensity imaging of Z-stacks. The cells
were manually counted. A total of 4 replicates were used per sample.
stem cell
line (ASC/TERT1) (Evercyte) was transfected with green fluorescent
protein (GFP) to obtain permanently transfected green-labeled cells
following a previously established protocol.22 (link) ASCs-GFP were cultured in endothelial cell growth (EGM-2) media
(Lonza) supplemented with 10% fetal bovine serum (Sigma). The cells
were maintained in an incubator (high humidity, 5% CO2,
37 °C), and the medium was changed every second day. To enable
printing in the presence of living cells, the cells were trypsinized
with 0.5% trypsin–EDTA and resuspended at 1 x 106 cells/mL in 7.5 and 10 w/v % RCPhC1-NB/SH solution supplemented
with 2 mol % DAS with respect to the photo-cross-linkable functionalities.
The solution was pipetted into a silicone mold in the glass bottom
methacrylated Ibidi dishes. Cubes of 200 μm × 200 μm
× 200 μm were printed using a writing speed of 1000 mm/s
and laser powers of 50, 70, and 100 mW. The proliferation rate was
determined by counting the cells in the cubes after days 1, 3, and
6, and the data were normalized to day 1. To this end, ImageJ software
was used to assess maximum intensity imaging of Z-stacks. The cells
were manually counted. A total of 4 replicates were used per sample.
6
Differentiating hiPSCs into Endothelial Cells
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The endothelial cell differentiation of DS hiPSC lines were adapted from Lian et al. (2014) (link) and Patsch et al. (2015) (link). In brief, two days before differentiation, hiPSCs were dissociated into a single cell suspension using TrypLE Express (ThermoFisher). The cells were resuspended in Essential 8 media with 10 uM Rock inhibitor Y-27632 (Tocris Bioscience) and seeded at 40,000 cells/well in a vitronectin-coated 12-well plate. Cells were fed with Essential 8 media the next day. On Day 0 and Day 1, the media was replaced with LaSR media as described in Lian et al. (2014) (link) supplemented with 8 uM CHIR99021 (Tocris Bioscience). On Day 2 and 4, the media was replaced with LaSR media supplemented with 2 uM CHIR99021. On Day 5, cells were dissociated using TrypLE Express and enriched for CD34+ endothelial progenitor cells using the CD34 MicroBead Kit (Miltenyi Biotec). Purified cells resuspended in EGM-2 media (Lonza) supplemented with 25 ng/mL VEGF165 (PEPROTECH). Cells were seeded onto Collagen I coated plates (50–100 ng/mL in 0.02 M acetic acid) and expanded for downstream experiments. Samples included all transgenic lines with or without dox and one isogenic disomic and trisomic line as a dox control. Three independent differentiations were performed.
7
Culturing Human Umbilical Vein Endothelial Cells
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HUVECs were obtained from the Yale School of Medicine, Vascular Biology and Therapeutics Core facility. Cells were cultured in EGM-2 media (Lonza) with 10% fetal bovine serum (FBS), penicillin/streptomycin and glutamine (2.8 mM) in a 37 °C incubator with 5% CO2 supply.
8
Isolation and Characterization of Pediatric Chylothorax-derived Lymphatic Endothelial Cells
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Chylous fluid samples were collected from pediatric patients who underwent cardiac surgery to correct structural congenital cardiac deformities (Table 1 ) with postoperative chylothorax fluid defined as: ≥80% lymphocyte count, and/or chylomicron positivity, and chyothorax fluid triglyerides (TG) > 50% serum TG levels (Columbia University IRB AAAQ6902). Patients with chromosomal anomalies were excluded.
Cells were depleted of CD133+ cells by magnetic bead isolation (Miltenyi) as described.18 (link) Nonadherent immune cells were removed after seeding and adherent CD133-negative cells were expanded and characterized by quantitative RT-PCR (qRT-PCR) and fluorescent activated cell sorting (FACS). HdLECs, isolated from neonatal dermis using CD31+ bead selection and live cell sorting for PODOPLANIN, served as normal controls.19 (link) pcLECs and HdLECs were maintained on fibronectin-coated plates in EGM-2 media (Lonza) supplemented with 18% FBS or EGM-MV2 media (Lonza), respectively.
Cells were depleted of CD133+ cells by magnetic bead isolation (Miltenyi) as described.18 (link) Nonadherent immune cells were removed after seeding and adherent CD133-negative cells were expanded and characterized by quantitative RT-PCR (qRT-PCR) and fluorescent activated cell sorting (FACS). HdLECs, isolated from neonatal dermis using CD31+ bead selection and live cell sorting for PODOPLANIN, served as normal controls.19 (link) pcLECs and HdLECs were maintained on fibronectin-coated plates in EGM-2 media (Lonza) supplemented with 18% FBS or EGM-MV2 media (Lonza), respectively.
9
Isolation and Culture of Endothelial Cells
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HDMECs were cultured in MV2 media with supplements (Promocell). MBECs were isolated from mice between 1 and 3 wk of age and cultured on tissue culture plates coated with 20 μg/ml FN in EGM2 media (Lonza) without passaging; 4 µg/ml puromycin (Sigma-Aldrich) was included in the media for 2 d to eliminate contaminating cell types. MLECs were isolated from mice between 1 and 2 mo of age by magnetic-activated cell sorting (MACS) with PECAM1 and ICAM2 antibodies (BD). MLECs were cultured on tissue culture plates coated with 10 µg/ml FN in DMEM-GlutaMAX supplemented with 20% FBS, nonessential amino acids (Life Technologies), and ECGS (Sigma-Aldrich). Cells were stimulated with 50 ng/ml VEGF165 (PeproTech; for HDMEC) or VEGF164 (for MLECs or MBECs) for the indicated times. In some experiments, HDMECs were incubated with inhibitors dissolved in DMSO or the same concentration of DMSO 30 min before VEGF165 stimulation. We used the following inhibitors: 10 µM Imatinib (Cambridge Bioscience), 10 µM PP2 (Sigma-Aldrich), and 0.1 µM PTK/ZK (Vatalanib; Selleckchem).
10
Angiogenesis Assay on Basement Membrane
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A 96-well plate was coated with basement membrane extract (50μL/well, Trevigen Gaithersburg, MD) 30 minutes prior to cells being plated on their surface at 20,000 cells per well. Cells were then incubated for 5 hours in EGM-2 media (Lonza Allendale, NJ) with 10% fetal bovine serum. Following incubation, cells were stained with Calcein AM (Fisher Scientific, Pittsburg, PA) and their ability to form capillary-like networks was measured. An EVOS microscope and Image PRO software was used to quantify tube formation.
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