and reagents including NaOH, cefazolin, (3-aminopropyl) triethoxysilane (APTES), HUVEC culture media, BCA Protein Assay kit 23225, Human VEGF 165, fetal bovine serum (FBS), Geltrex matric, and PBS were purchased from Sigma-Aldrich (MO, USA). Polydimethylsiloxane (PDMS) was purchased from Dow (MI, USA). 3D-printing resins were obtained from Stratasys (MN, USA). Recombinant mouse VEGF (VEGF 164) was purchased from Biolegend (CA, USA) and anti-CD31 antibody (ab56299), anti-MMP9 antibody (ab38898), were purchased from Abcam (Cambridge, MA). gGoat antirat IgG (H+L) secondary antibody, Alexa Fluor 488, and goat antirabbit IgG (H+L) secondary antibody, Alexa Fluor 594 were purchased from Thermo Fisher Scientific (Waltham, MA).
Human vegf165
Manufactured by Merck Group
Sourced in United States, United Kingdom
Human VEGF165 is a recombinant protein that represents the 165-amino acid isoform of Vascular Endothelial Growth Factor (VEGF). VEGF is a key regulator of angiogenesis and vascular permeability. This product can be used for cell culture, biochemical, and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Elisa plate, by Corning (2 mentions)
Sunrise spectrophotometer, by Tecan (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Polydimethylsiloxane pdms, by Dow (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Adenosine, by Merck Group (1 mentions)
Cgs15943, by Bio-Techne (1 mentions)
Ebm 2, by Lonza (1 mentions)
Bevacizumab, by Genentech (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Biacore t200, by GE Healthcare (1 mentions)
Amine coupling kit, by GE Healthcare (1 mentions)
6 protocols using human vegf165
1
3D-Printed Vascularized Tissue Model
2
Adenosine and VEGF Signaling in HUVECs
3
Evaluating DARC in Retinal Angiogenesis Model
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A pilot study was also performed on three New Zealand White rabbits to assess DARC using an established model of retinal angiogenesis [21 ]. All experiments were designed and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and under protocols approved by the UK. Home Office. Briefly, 2.5 kg rabbits were anesthetized with 50 mg/kg intramuscular injection of ketamine hydrochloride and 10 mg/kg xylazine, respectively. Pupils were dilated with 2.5% phenylephrine and 1% tropicamide. 50 ul containing 1 ug of humanVEGF165 (Sigma, UK) dissolved in 0.1% bovine serum albumin (Sigma UK) was injected intravitreally into the left eye only of each animal. Forty-eight hours later, under general anesthesia, rabbits received intravenous Anx776 (0.2 mg) and 1% sodium fluorescein and underwent DARC and 448 imaging at 40 min using high-resolution ICGA and FFA modes of the HRA-Spectralis. Three masked observers performed manual counts on DARC images.
4
Quantifying Aflibercept Binding to VEGF
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96-well ELISA plates (Corning-Costar) were coated with 0.2 µg/ml human VEGF165 (Sigma-Aldrich) diluted in PBS (Sigma-Aldrich). After overnight incubation at 4 °C, plates were blocked with PBS containing 4% skimmed milk (S) (Sigma-Aldrich) for 1 hour at room temperature and washed 4 times with PBS containing 0.05% Tween-20 (T) (Sigma-Aldrich). Serial dilutions of collected aflibercept samples (1000.0–0.5 ng/ml) in PBS/S/T were added to the plates followed by incubation at room temperature for 1.5 hours. Plates were then washed as described above, before an alkaline phosphatase-conjugated goat polyclonal antibody directed towards human IgG Fc (Sigma-Aldrich), diluted (1:5000) in PBS/S/T, was added and incubated at room temperature for 1.5 hours. The plates were washed as above before 100 µl of the p-nitrophenylphospate substrate (Sigma-Aldrich), diluted to 10 µg/ml in diethanolamine buffer, was added. The absorbance was measured at 405 nm using a Sunrise spectrophotometer (TECAN).
5
Surface Plasmon Resonance Binding Analysis of Aflibercept to VEGF
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SPR was performed using a Biacore T200 instrument (GE Healthcare) with CM5 sensor chips coupled with human VEGF165 (Sigma-Aldrich) (~300 resonance units (RU)) using amine-coupling chemistry as described by the manufacturer. Briefly, the coupling was performed by injecting 5 μg/ml of VEGF diluted in 10 mM sodium acetate, pH 4.5 (GE Healthcare) using the amine-coupling kit (GE Healthcare). HBS-EP pH 7.4 buffer (0.01 M HEPES, 0.15 NaCl, 3 mM EDTA 0.005% surfactant P20) was used as both dilution and running buffer. Binding analysis was performed by injecting 100 nM of each aflibercept sample over the immobilized surface using a flow rate of 50 µl/min and 10 mM NaOH was used to regenerate the CM5 surface. The sample compartment was kept at 4 °C while the binding analysis was performed at 25 °C. The binding data were zero adjusted and the maximum binding responses of the individual injections were normalized using the BIAevaluation software version 4.1.
6
Aflibercept-FcRn Binding Assay
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96-well ELISA plates (Costar) were coated with 0.5 µg/ml human VEGF165 (Sigma-Aldrich) and blocked before serial dilutions of collected aflibercept samples were added as above. Plates were washed with PBS/T (pH 7.4) or 67 mM phosphate buffer (pH 5.5), before 1.0 µg/ml of GST-tagged receptor FcRn35 (link), diluted in pH 7.4 or pH 5.5 buffer, was added and incubated at room temperature for 1.5 hours. The plates were washed as above before a horseradish peroxidase-conjugated goat polyclonal antibody directed towards GST (Rockland Immunochemicals Inc), diluted (1:8000) in pH 7.4 or pH 5.5 buffer, was added and incubated at room temperature for 1.5 hours. After washing as above, bound receptor was detected using the tetramethylbenzidine substrate (Calbiochem) and the reaction was stopped by adding 50 µl 1 M HCl. The absorbance was measured at 450 nm using a Sunrise spectrophotometer (TECAN).
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