Human Umbilical Vein Endothelial Cells (HUVECs) were isolated from human umbilical cords following established protocols (49 (link)). The cells used in experimental replicates were isolated from different donors. Cells were grown in EGM™-2 Endothelial Cell Growth Media (Lonza) (including all supplements provided by bullet kits) on culture dishes coated with rat tail type I collagen (Corning). Human Retinal Microvascular Endothelial Cells (HRMECs) were purchased from Cell Systems. Cells were maintained on fibronectin (Sigma) coated plates (Millipore) and in EGM™-2 Endothelial Cell Growth Media (Lonza). 293T cells were acquired from ATCC and maintained in High Glucose DMEM (Gibco) with 10% Heat-Inactivated Fetal Bovine Serum (HI-FBS) and 0.01% penicillin-streptomycin. Unless otherwise noted, cells were cultured under standard conditions in a humidified incubator at 37°C, 5% CO2.
Egm 2 endothelial cell growth media
Manufactured by Lonza
EGM-2 Endothelial Cell Growth Media is a complete culture medium designed for the growth and proliferation of human endothelial cells. The media contains essential nutrients, growth factors, and other components required for the optimal growth and maintenance of endothelial cell lines in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fluorescent polystyrene beads, by Bangs Laboratories (1 mentions)
Fitc dye, by Merck Group (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Syringe pump, by Chemyx (1 mentions)
Endothelial cell basal medium 2, by Lonza (1 mentions)
Hmvec, by Lonza (1 mentions)
El4 cells, by American Type Culture Collection (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Anti ng2, by Merck Group (1 mentions)
Anti mmp14, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Low glucose dmem, by Thermo Fisher Scientific (1 mentions)
Anti pdgfrβ, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Anti mmp2, by Abcam (1 mentions)
8 protocols using egm 2 endothelial cell growth media
1
Isolation and Culture of Human Endothelial Cells
2
In Vitro Endothelial Cell Constriction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FITC dye (1 μM, Sigma-Aldrich) with Endothelial cell Growth Media (EGM-2, Lonza) was introduced into the cell culture channel and imaged using a fluorescence microscope (Nikon Eclipse Ti). Channel constrictions were realised by pumping-in air into the pneumatic channel using a syringe pump (Chemyx, Inc.) while simultaneously monitoring the FITC fluorescence-linescan intensity. The fluorescence linescan intensity drops as the channel constriction increases, which was used to measure the percentage of channel constriction (Fig. S1, supplementary material ). A 3D reconstruction of the stenosis region was performed from the FITC fluorescence images (at different channel constrictions) using ImageJ (NIH) for better visualization of the 3D-stenosis. Confocal imaging (LSM 800, Carl Zeiss) of the FITC-loaded channels was performed to visualize the 3D stenosis. Flow characterization was performed by pumping 10 μm fluorescent polystyrene beads (Bangs Laboratories) through the culture channel. Time-lapse images were obtained to calculate the beads rolling velocities which were processed using ImageJ.
3
Endothelial Cell Cultivation and Starvation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVEC-I and MVEC were provided by Dr. A. Kelekar and Dr. J. Hall respectively (University of Minnesota). Primary HUVEC cultures were purchased from Neuromics (Edina, MN). Endothelial cells were cultured in Endothelial cell Growth Media EGM-2™ (Lonza). For growth factor starvation, Endothelial cell Basal Medium-2™ (Lonza) was used. Experiments were performed on primary HUVEC obtained from two different batches and MVEC used in real-time experiments were procured from a single batch. LLC cells were obtained from ATCC (Manassas, VA) and grown in RPMI 1640 medium supplemented with 10% FBS, 1% glutamine, 1% Penicillin-Streptomycin and 1% sodium pyruvate.
4
Culturing Endothelial and Cancer Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human umbilical vein endothelial cells from pooled donors (HUVEC, Lonza Walkersville Inc., Walkersville, MD, #C2519AS), passage 4-8, were cultured on 0.1% gelatin-coated plates in EGM-2 endothelial cell growth media (Lonza Walkersville Inc., Walkersville, MD, #CC3162). Primary human lung microvascular endothelial cells from single donor (HMVEC, Lonza Walkersville Inc., Walkersville, MD, #CC-2527), passage 3-6, were cultured on 0.1% gelatin coated plates in EGM-2 microvascular cell growth media (Lonza Walkersville Inc., Walkersville, MD, #CC3202). EL4 cells were purchased from ATCC (Manassas, VA, #TIB-39) and maintained in high glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT, #SH30243.01) supplemented with 10% fetal bovine serum (FBS, Omega Scientific, Inc., Tarzana, CA, #FB-02). The GL261 cell line [43] (link) was a gift from Dr. Santosh Kesari (UCSD) and was maintained in high glucose DMEM supplemented with 10% FBS. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO 2 .
5
Human Brain Vascular Pericyte Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human brain vascular pericytes (HBVP) (ScienCell) were maintained in 1× Low Glucose DMEM (Invitrogen) with 10% fetal bovine serum and 1× Pen-Strep (Invitrogen). Human umbilical vein endothelial cells (HUVEC) were isolated as described61 (link). HUVEC were grown in EGM-2 Endothelial Cell Growth Media (Lonza Group). Knockdown cell lines were made using stable lentiviral infection (see supplemental information ). RNA was collected from cultured cells using the RNeasy Mini Kit (Qiagen). Protein lysates was collected 4 days post-infection in TENT lysis buffer containing 1× HALT protease inhibitor cocktail (Thermo Fisher). The following antibodies were used for western blotting in 2% milk, 2% BSA, 0.1% Tween in 1× PBS: anti-NG2 (Millipore), anti-PDGFR-β (Cell Signaling), anti-MMP14 (Abcam), anti-MMP2 (Abcam), anti-vinculin (Sigma), and HRP-conjugated secondary antibodies (Sigma).
6
Angiosarcoma and SVR Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SVR cells were obtained from ATCC and were maintained in Dulbecco's Modified Eagle Medium (DMEM) + 5% FBS + 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). ASM-1 angiosarcoma cells were generously provided by Dr. James Kirkpatrick (Johannes Gutenberg-Universität Mainz, Germany)14 . HAMON angiosarcoma cells were generously provided by Dr. Riichiro Abe (Hokkaido University, Japan)15 (link). ASM-1 and HAMON cells were maintained in EGM-2 endothelial cell growth media (Lonza). All cells were screened for mycoplasma and experiments were performed at 60–80% confluence.
7
Cell Culture and Transfection Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 (Stratagene) and HeLa (ATCC) cells were cultured in DMEM with 10% fetal bovine serum (FBS). HUVECs and VSMCs (Lonza) were cultured in EGM-2 Endothelial Cell Growth Media or SMGM-2 Smooth Muscle Cell Growth Media (Lonza), respectively. All cells were maintained in sub-confluent densities to allow cell division throughout the course of the experiments. Experiments using HUVECs and VSMCs were carried out in cells between 3 and 7 passages. Cells were plated in 24-well plates (HEK293, HeLa, and VSMC, 5 × 104/well; HUVEC, 1 × 105/well) on the day prior to the first transfection. miRIDIAN miRNA mimics and hairpin inhibitors (Dharmacon) were transfected at a final concentration of 200 nM using DharmaFECT 4 Transfection Reagent (Dharmacon). Lipofectamine 2000 (Thermo Fisher Scientific) was used for mRNA transfections. Media were changed after 4 h to remove transfection reagent from HUVECs, VSMCs, and HeLa cells. Cells were collected 24 h following mRNA transfection.
8
Evaluating HUVEC Viability under Hydrogel Degradation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!