Human umbilical vein endothelial cells (HUVECs; passage 4–6; C0035C (newborn, single donor) Thermo Fisher Scientific, USA) were grown to confluency in Medium 200 (Thermo Fisher Scientific, USA) supplemented with large vessel endothelial supplement (LVES 50×; Thermo Fisher, USA) at 37 °C/5% CO2. Cells were seeded at 5000 cells per 100 μl/per well onto black, clear bottomed 96 well plates (Grenier Bio-One, 655090) and left to grow at 37 °C/5% CO2 for 24 h. The next day, the plating medium was replaced with 150 μl per well of Medium 200 with 0.1% serum for a further 24 h at 37 °C/5% CO2. On day 3, cells were stimulated in triplicate wells for 24 or 48 h at 37 °C/5% CO2 with 3 nM or 30 nM VEGF165a (R and D Systems) or VEGF165a-TMR in Medium 200/0.1% serum. At the end of the incubation period, cells were washed with PBS (100 μl/well), fixed using 3% PFA/PBS (20 min at room temperature) and nuclei stained using H33342 (2 mg/ml; 15 min at room temperature). Nuclei were imaged at 4 sites per well using a IX Micro widefield platereader (Molecular Devices, USA) fitted with a 4× objective using a DAPI filter with 25 ms exposure time. Nuclei were counted using a granularity algorithm (MetaXpress, Molecular Devices, USA). Data were normalised to show fold increases in proliferation compared to vehicle treatment and expressed as mean ± S.E.M.
Vegf165a
Manufactured by R&D Systems
Sourced in United Kingdom
VEGF165a is a recombinant human protein that represents the predominant isoform of vascular endothelial growth factor (VEGF). VEGF165a is a key regulator of angiogenesis and vasculogenesis and plays a critical role in the growth and development of blood vessels.
Automatically generated - may contain errors
Lab products found in correlation
Topcount plate reader, by PerkinElmer (4 mentions)
One glo luciferase reagent, by Promega (3 mentions)
Vegf165b, by R&D Systems (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Pdgf bb, by R&D Systems (1 mentions)
Recombinant human egf, by R&D Systems (1 mentions)
Medium 200, by Thermo Fisher Scientific (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Ici 118 551, by Bio-Techne (1 mentions)
Cgp 12177, by Bio-Techne (1 mentions)
Fugene hd, by Promega (1 mentions)
Imagexpress micro widefield platereader, by Molecular Devices (1 mentions)
Versene, by Lonza (1 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Reluc2p, by Promega (1 mentions)
10 protocols using vegf165a
1
Endothelial Cell Proliferation Assay
2
Comprehensive Signaling Pathway Analysis
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Recombinant human EGF and PDGF-BB were from R&D Systems and recombinant human VEGF165a was from Life technologies. Research antibody targeting EGF was from Cell Signaling and research antibodies targeting PDGF-BB, PDGFRβ and VEGF165a were from R&D systems. The therapeutic antibodies Cetuximab, Panitumumab, Bevacizumab and Trastuzumab were from Myoderm and DY605-NHS-ester was obtained from DYOMICS GmbH.
3
GPCR Activation Monitoring Protocol
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All materials were purchased from Sigma-Aldrich (Gillingham, UK) unless otherwise stated. FuGENE-HD, ONE-Glo™ luciferase and HaloTag® AF488 ligand were purchased from Promega Corporation (Wisconsin, USA). Opti-MEM was purchased from Thermo-Fisher Scientific (Massachusetts, USA). SNAP-Tag® AlexaFluor 647 (AF647) and AlexaFluor 488 (AF488) were purchased from New England BioLabs (Massachusetts, USA). VEGF165a was purchased from R&D Systems (Abingdon, UK). VEGF165a-TMR was produced as described by Kilpatrick et al. (2017) (link). BODIPY-CGP12177-TMR (Baker et al., 2003 (link)) was purchased from Molecular Probes (Oregon, USA). CGP12177 and ICI118551 were purchased from Tocris Bioscience (Bristol, UK). The cDNA construct expressing β-arrestin2-Venus-YFP was provided by Dr KDG Pfleger and generated as described in Kocan et al. (2008) (link).
4
VEGF Stimulation of HUVECs Imaging Assay
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HUVECs (passage 4-9) were seeded at 5,000 cells/well in black flat-bottomed 96-well plates (Greiner Bio-One, 655090) in 10% LVES/Medium 200. Following 24 hours of cell growth at 37°C/5% CO2, plating medium was replaced with Medium 200 containing 0.1% serum for 24 hours. Cells were then stimulated with commercially available VEGF121a or VEGF165b (R&D Systems), VEGF121a-TMR or VEGF165b-TMR (Promega Corporation, USA) at 0.3nM, 3nM or 30nM (in 0.1% serum/medium), or positive control 3nM VEGF165a (R&D Systems). Following 48 hour stimulation at 37°C/5% CO2, cells were washed with 100μl/well PBS, fixed with 3% PFA/PBS (20 minutes, room temperature ) and nuclei stained with 2mg/ml H33342 (15 minutes, RT). Nuclei were imaged using an ImageXpress Micro widefield platereader (Molecular Devices, USA) with a 4x objective using a DAPI filter (4 sites per well, 25ms exposure time).
5
VEGFR2-Mediated Luciferase Transcriptional Assay
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HEK293T cells stably expressing wild‐type or tyrosine phosphorylation‐deficient HaloTag‐VEGFR2, as well as NFAT‐RE‐luc2P, were grown to 70–80% confluency. Cells were seeded at 25,000 cells per well in white 96‐well plates pre‐coated with poly‐D‐lysine. Following 24 hr at 37%/5% CO2, cell culture media were replaced with serum‐free DMEM for another 24 hr. Cells were then stimulated with increasing concentrations of VEGF165a (R&D Systems) or vehicle (serum‐free DMEM/0.1% BSA). Following stimulation for 5 hr at 37%/5% CO2, media were replaced with 50 μl per well assay buffer and 50 μl per well ONE‐Glo Luciferase reagent (Promega Corporation, USA). Cells were incubated for 5 min to allow luciferase to react with the added reagent and for the background luminescence to subside, and then luminescence emissions were measured using a TopCount platereader (PerkinElmer, UK). Data were normalised to their respective vehicle (0%) and response of wild‐type HaloTag‐VEGFR2 to 10‐nM VEGF165a (100%) per experiment. Data were pooled from five independent experiments with duplicate wells.
6
Quantifying VEGF Protein Signaling
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VEGF165a and VEGF165b were obtained from R&D systems (Abingdon, UK). Vandetanib, pazopanib, cediranib and sorafenib were supplied by Sequoia Research Products (Pangbourne, UK). The ONE-Glo™ Luciferase Assay System was obtained from Promega Corporation (Madison, WI, USA). Versene was obtained from Lonza (Basal, Switzerland). G418 was purchased from Life Technologies (Paisley, UK). All other chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK).
7
VEGFR2-Mediated NFAT Transcriptional Assay
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HEK293T cells stably expressed HaloTag-VEGFR2 or NanoLuc-VEGFR2, as well as ReLuc2P, a Firefly luciferase reporter gene inserted downstream of the NFAT reporter to monitor NFAT-induced gene transcription (Promega Corporation, USA). Cells were grown to 70-80% confluency and seeded in DMEM containing 10% Fetal Calf Serum at 25,000 cells per well in white 96-well plates (Greiner Bio-One, 655089) pre-coated with poly-D-lysine (0.1mg/ml in PBS). Following 24 h of cell growth at 37°C/5% CO2, plating medium was replaced with serum free DMEM for a further 24 h. Cells were stimulated in duplicate wells with increasing concentrations of VEGF165a (R&D Systems) or vehicle (serum free DMEM containing 0.1% BSA) for 5 h at 37°C/5% CO2. Assay medium was then replaced with 50μl/well serum free DMEM and 50μl/well One-Glo Luciferase reagent (Promega Corporation, USA) and equilibrated for 5 min to enable the reagent to react with luciferase and allow background luminescence to subside. Luminescence was then measured by a TopCount platereader (Perkin Elmer, UK). Data were normalised to vehicle (0%) and response to 10nM VEGF165a (100%) in each experiment and expressed as mean ± SEM. (5 independent experiments). Potency (EC50) values were derived as described previously (Kilpatrick et al., 2017 (link)).
8
VEGFR2 Signaling Pathway Activation
9
HUVEC Proliferation Assay with VEGF and Isoprenaline
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HUVECs (passage 4-8) were seeded at 5,000 cells/well in black flat-bottomed 96-well plates (Greiner Bio-One, 655090) in 10% LVES/Medium 200. Following 24 h of cell growth at 37°C/5% CO2, plating medium was replaced with Medium 200 containing 0.1% serum for 24 h. Cells were then stimulated with VEGF165a (R&D Systems) at 3nM (in 0.1% serum/medium), Isoprenaline (100nM or 10μM) or both ligands simultaneously in the presence or absence of the receptor tyrosine kinase inhibitor cediranib (1μM; Sequoia Research Products, UK). Cediranib (1μM) was also used alone as a negative control. Following 48 h stimulation at 37°C/5% CO2, cells were washed with 100μl/well PBS, fixed with 3% PFA/PBS (20 min, room temperature) and nuclei stained with 2mg/ml H33342 (15 min, room temperature). Nuclei were imaged using an ImageXpress Micro widefield high content imaging system (Molecular Devices, USA) with a 4x objective using a DAPI filter (4 sites per well, 25ms exposure time).
10
VEGFR2-Mediated Luciferase Assay
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HEK293T-NFAT-ReLuc2P cells stably expressed LgBiT-VEGFR2, HiBiT-VEGFR2 or SmBiT-VEGFR2. Cells were seeded at 25,000 cells/well in white 96-well plates pre-coated with poly-D-lysine in DMEM containing 10% FBS. Following incubation for 24 hours at 37%/5% CO2, medium was replaced with serum-free DMEM and cells were incubated for a further 24 hours. On the day of experimentation, medium was replaced with serum-free DMEM containing 0.1% BSA. Cells were stimulated with increasing concentrations of VEGF165a (R&D Systems) for 5 hours at 37%/5% CO2. Medium was replaced with 50 μl/well serum-free DMEM/0.1% BSA and 50 μl/well ONE-Glo Luciferase reagent. Following a 5 minute delay to allow reagent to react with luciferase and background luminescence to subside, luminescence emissions were measured using a TopCount platereader (Perkin Elmer, UK).
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