HUVECs were obtained from Promocell. Three independent lines were used for all experiments. HUVECs were cultured in Endothelial Cell Growth Medium MV (Promocell). To knock-down JCAD, 2 unique siRNAs (Qiagen, SI04210136, SI04344704) were reverse transfected into the cells using RNAiMAX transfection reagent (Life Technologies, 10514953), LATS2 knockdown was achieved using a Dharmacon Smart pool of 4 siRNAs (GE Life Sciences, L-003865-00-0005). HAECs were cultured in EGM-2 media (Lonza, CC-3156) and JCAD knockdown achieved with a Dharmacon Smart pool of 4 siRNAs (GE Life Sciences, L-026476-02-0005), also transfected using RNAiMAX.Thp-1s were cultured in RPMI (Corning, 10-040-CVR) supplemented with 10% FBS. HEK293A cells were cultured in DMEM with 10% FBS. Drug treatments were carried out in full Endothelial media, for 1 hour treatment time, TRAP6 (Sigma-Aldrich, T1573) was used at 1 µM and Y-27632 (Stemcell Technologies, 72304) at 10 µM.
Endothelial cell growth medium mv
Manufactured by PromoCell
Sourced in Germany
Endothelial Cell Growth Medium MV is a serum-free, low-protein medium designed for the cultivation of primary human endothelial cells. The medium contains the necessary growth factors and supplements to support the proliferation and maintenance of endothelial cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Dmem f12 media, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Egm tm 2 endometrial cell growth medium 2, by Lonza (3 mentions)
Hdmec, by PromoCell (3 mentions)
Huvecs, by PromoCell (2 mentions)
Hdbec, by PromoCell (2 mentions)
Hemec, by PromoCell (2 mentions)
Recombinant human cxcl12, by Thermo Fisher Scientific (2 mentions)
Hutmec, by PromoCell (2 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Haoec, by PromoCell (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ypd broth, by Thermo Fisher Scientific (2 mentions)
Trap 6, by Merck Group (1 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dharmacon smart pool of 4 sirnas, by GE Healthcare (1 mentions)
Y 27632, by STEMCELL (1 mentions)
23 protocols using endothelial cell growth medium mv
1
Endothelial Cell Knockdown Protocol
2
Knockdown of Endocytosis Regulators in HDBECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following control siRNA and siRNA that targets caveolin 1 (CAV1), clathrin heavy chain (CLTC), or c-ABL (ABL1) were purchased from GE healthcare (Pollards Wood, UK): Accell human CAV1 siRNA-SMART pool (Cat. E-003467-00-0005), Accell human CLTC siRNA-SMART pool (Cat. E-004001-00-0005), Accell human ABL1 siRNA-SMART pool (Cat. E-003100-00-0005), and Accell Nontargeting Pool (Cat. D-001910-10-05). HDBECs (PromoCell) were treated with each siRNA following the manufacturer’s protocol.
Briefly, HDBECs were cultured in endothelial cell growth medium MV (PromoCell) with 5% serum concentration, at 37 °C in 5% CO2, until reaching 40–50% confluency. The medium was then exchanged to siRNA containing MV medium with 2.5% serum concentration, and cultured for another 3 days at 37 °C in 5% CO2. Down regulation of each mRNA expression was verified at this time point by reverse transcription polymerase chain reaction. Subsequently, the medium was again exchanged to MV medium with 5% serum concentration, and cells were cultured for additional 3 days at 37 °C in 5% CO2. After that, immunohistochemistry to check the down regulation of protein expression of caveolin 1 and clathrin, and the IgG endocytosis assay were performed as mentioned.
Briefly, HDBECs were cultured in endothelial cell growth medium MV (PromoCell) with 5% serum concentration, at 37 °C in 5% CO2, until reaching 40–50% confluency. The medium was then exchanged to siRNA containing MV medium with 2.5% serum concentration, and cultured for another 3 days at 37 °C in 5% CO2. Down regulation of each mRNA expression was verified at this time point by reverse transcription polymerase chain reaction. Subsequently, the medium was again exchanged to MV medium with 5% serum concentration, and cells were cultured for additional 3 days at 37 °C in 5% CO2. After that, immunohistochemistry to check the down regulation of protein expression of caveolin 1 and clathrin, and the IgG endocytosis assay were performed as mentioned.
3
Endometrial Cell Culture and Angiogenesis Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human endometrial epithelial Ishikawa cells (ATCC) and CRL4003 cells, kindly gifted from the laboratory of Professor Haeng-Seok Song of CHA University, were maintained in DMEM/F12 media (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Gibco, Grand Island, NY, USA) as previously described32 (link). For tube formation and sprouting assays, Human Umbilical Vein Cells (HUVEC, ATCC) and Human Endometrial Microvascular Endothelial Cells (HEMEC, PromoCell) were maintained in EGM-2 endometrial cell growth medium 2 (Lonza) and Endothelial Cell Growth Medium MV (PromoCell), respectively. Human recombinant CXCL12 (100 ng/ml, Peprotech, USA) was used for further analyses.
4
Endothelial Cell Signaling Pathway Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-p-p65 antibody, Gö6976, heparin, PMA, Röttlerin, and PKC-δ siRNA were purchased from Sigma-Aldrich (St.Louis, MO). Bay 11-7082, Bay 61-3606, piceatannol, and caffeic acid phenethyl ester (CAPE) were purchased from Cayman Chemicals (Ann Arbor, MI). Lipofectamine 3000 reagent was acquired from Invitrogen Life Technologies (Gaithersburg, MD). Antibody specific for Thy-1, and p-PKC-δ was purchased from Cell Signaling Technology (Danvers, MA). Anti-Syk, p65, PKC-δ, and G3PDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for p-Syk was purchased from EMD Millipore (Temecula, CA). Dual-Luciferase assay kit was purchased from Promega (Madison, WI). Penicillin and streptomycin were purchased from HyClone (South Logan, Utah). Endothelium cell growth factor (ECGS) was purchased from Biomedical Technologies (Stoughton, MA). Fetal calf serum (FCS), Medium 199 (M199), and GlutaMAX Supplement were obtained from GIBCO (Grand Island, NY). Endothelial Cell Growth Medium MV was purchased from PromoCell (Heidelberg, Germany).
5
Cytotoxicity of 4-MU on Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the cytotoxicity of different doses of 4-MU on endothelial cells, we performed a LDH assay by means of the Cytotoxicity Detection KitPLUS (Roche, Mannheim, Germany) according to the manufacturer's instructions. For this purpose, human dermal microvascular endothelial cells (HDMEC; PromoCell, Heidelberg, Germany) were cultured in Endothelial Cell Growth Medium MV (PromoCell) at 37°C under a humidified 95% to 5% (v/v) mixture of air and CO2 until they reached confluence. The cells were then stimulated with 1, 2 and 4mM 4-MU (n = 4 each) or 0.9% NaCl as vehicle control (n = 4). After 24h, 100μL of reaction mix per 100μL medium was added to each well. After 10min at room temperature in the dark, the reaction was stopped by addition of 50μL stop solution. Absorption was then measured at 492nm with 620nm as reference using a microplate reader and corrected to blank values (wells without cells). As a positive control cells were treated with a lysis buffer and subjected to the same procedure. The concentrations tested were chosen according to previous studies [21 (link),25 (link)].
6
Quantifying IgG Endocytosis in HDBECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HDBECs were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial cell growth medium MV (PromoCell) at 37 °C in 5% CO2 until reaching 70–80% confluency. For the IgG endocytosis assay, cells were incubated in the presence of A594-conjugated human IgG (Cat. 009-580-003, 10 μg ml−1; Jackson ImmunoResearch) for 1 h. To block dynamin function, cells were treated with dynasore (100 μg ml−1; Santa Cruz Biotechnology, Dallas, TX) for 30 min. To block caveolae-mediated endocytosis, cells were cultured with nystatin (50 μg ml−1; Sigma Aldrich) for 30 min. To evaluate the quantity of engulfed IgG in each cell, the cytoplasmic area of cells in a high-power field (×40) was manually circumscribed using ImageJ58 (link), and the MFI of the signals from A594-IgG was measured. ΔA594-IgG was calculated by subtracting the background MFI.
7
Endothelial Cell Internalization of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A semi-immortalized human bone marrow derived endothelial cell line (BMEC-1)62 (link) was cultured in Endothelial Cell Growth Medium MV (Promocell) supplemented with 10% fetal bovine serum (Cedarlane, Burlington, Canada) and maintained at 37 °C under 5% CO2.
EV internalization assays with BMEC-1. Purified EVs were labelled with PKH67 red fluorescent labelling kit (Sigma-Aldrich) and incubated with BMECs for 12 h before washing cells three times to remove unbound EVs. For uptake inhibition experiments endothelial cells were co-incubated with different compounds, as described in Fig.5 . Cells were fixed, permeabilized and stained for F-actin with CF594 (Biotium) and with the nuclear dye DAPI (Sigma-Aldrich). Leica TCS SP5. Epifluorescence was performed on a Axiovert 200 M microscope (Carl Zeiss. Germany) equipped with a camera (Orca charge-coupled device; Hamamatsu Photonics, Japan) using a 40 Plan-Neofluar (NA 1.3) oil immersion objective. Intensity analysis was performed with the Axiovision 4.6.3 software. Background subtraction was performed for each field. Confocal microscopy was performed on a Zeiss LSM 510 Laser Scanning Microscope using a 63 Plan-Neofluar water-immersion objective.
EV internalization assays with BMEC-1. Purified EVs were labelled with PKH67 red fluorescent labelling kit (Sigma-Aldrich) and incubated with BMECs for 12 h before washing cells three times to remove unbound EVs. For uptake inhibition experiments endothelial cells were co-incubated with different compounds, as described in Fig.
8
Culturing Human Endothelial and Angiosarcoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pooled human umbilical vein endothelial cells (HUVECs) were purchased from Pelobiotech (Frankfurt, Germany) and cultured in EGM-2 medium (Lonza; Basel, Switzerland). HUVECs were used between passage 1 and 5. HEK293 cells were obtained from the American Type Culture Collection (LGC Standards, Molsheim Cedex, France) and cultured in DMEM (Sigma) with 10% fetal bovine serum (FBS; Biochrom GmbH, Berlin, Germany), 2 mM L-glutamine (Gibco, Invitrogen, Life Technologies; Darmstadt, Germany), 100 IUml−1 penicillin, and 100 mg/ml streptomycin. Angiosarcoma cell lines ISO-HAS-B and AS-M were provided by Dr. Mikio Masuzawa and Dr. Ronald E. Unger and were cultured in Endothelial cell growth medium MV (PromoCell; Heidelberg, Germany).
9
Endothelial Cell Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human umbilical vein endothelial cells (HUVECs) (3 independent donors), human aortic endothelial cells (HAoECs), human pulmonary microvascular endothelial cells (HPMVECs) and human dermal microvascular endothelial cells (HDMVECs) (2 independent donors, respectively) were purchased from PromoCell (Heidelberg, Germany). HUVEC and HAoEC were cultivated in endothelial cell growth medium (PromoCell) with supplements including 2%/5% heat-inactivated fetal calf serum, respectively and used in passages 4 to 7. HPMVEC and HDMVEC were cultivated in endothelial cell growth medium MV (PromoCell) with supplements and 5% heat-inactivated fetal calf serum and used in passages 4 to 7. Cells were maintained at 37 °C, 5% CO 2 and 100% humidity until confluence. For the experiments, ECs were plated in 12 well plates at a seeding density of 1.5 × 10^5 cells/well or in 24 well plates at a seeding density of 8 × 10^4 cells/well. Except for immunofluorescence studies, cells were used for the experiment when it reached a desired confluency of 75–80%. The final molar concentration of serum proteins A1AT, hemopexin and albumin used for experiments were 9.62 μM, 8.47 μM and 7.52 μM respectively, corresponding to a concentration of 0.5 mg/ml in either 1 ml of medium/well for 12 well plates or 0.5 ml/well for 24 well plates.
10
Isolation of Primary Endothelial Cells
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!