Recombinant human VEGF (ThermoFisher Scientific, Waltham, MA, USA) and rat VEGF were resuspended in PBS and serially diluted. One microliter of solution was blotted on nitrocellulose membrane, blocked with Super Block (ScyTek Laboratories, Logan, UT, USA), and incubated with bevacizumab (250 μg/mL). After washing the membrane with tris-buffered saline, the membrane was incubated with a goat anti-human IgG horseradish peroxidase conjugated antibody (1:5000; Thermo Fisher Scientific). The blot then was incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and imaged.
Recombinant human vegf
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Recombinant human VEGF is a protein that plays a key role in angiogenesis, the process of new blood vessel formation. It is a potent stimulator of endothelial cell proliferation and migration, which are essential for the development of new blood vessels.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Matrigel, by BD (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Super block, by ScyTek Laboratories (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Total vegfr2, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Pperk1 2, by Cell Signaling Technology (1 mentions)
P vegfr2 tyr1175, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Acryloyl chloride, by Merck Group (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Santa Cruz Biotechnology (1 mentions)
Anti vegfr 1, by Santa Cruz Biotechnology (1 mentions)
29 protocols using recombinant human vegf
1
VEGF Binding Assay Protocol
2
Angiogenic Potential of HUVECs
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HUVECs were divided into five groups: Control, NaHS treated, NaHS treated + siRNA-NC, NaHS treated + siHIF-1α and VEGF treated. Matrigel (BD Biosciences; Becton, Dickinson and Company, San Jose, CA, USA) was diluted with FBS-free and growth factor-free M200 medium at a ratio of 1:1. Then, 50 µl of the mixture was placed into 96-well plates and incubated at 37°C for 30 min. Subsequently, a cell suspension containing 1×104 HUVECs were added into the 96-well plates coated with Matrigel. Next, 50 ng/ml of recombinant human VEGF (Gibco; Thermo Fisher Scientific, Inc.) was added to the medium as the positive control group (the VEGF group). Following culturing for 12 h, tube formation was observed under a light microscope at a magnification ×100 and total tube area was evaluated using ImageJ software (v.1.42).
3
Structural Modification and Evaluation of Asiatic Acid Derivatives
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AA was extracted from C. asiatica (L.) Urban and structurally modified in our laboratory to produce AA-PMe.20 (link) AA and AA-PMe were both dissolved in 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich Co, St Louis, MO, USA) and their structures are presented in Figure 1 . Endogenous alkaline phosphatase (EAP) staining was tested by a phosphatase substrate kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Recombinant human VEGF was purchased from Thermo Fisher Scientific. Primary antibodies for total Akt, pAkt-ser473, total ERK1/2, p-pERK1/2, total VEGFR2, and pVEGFR2-Tyr1175 were brought from Cell Signaling Technology (Danvers, MA, USA). GAPDH and tubulin antibody were from Abcam (Cambridge, UK).
4
In Vitro Wound Healing Assay for eMSCs
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For in vitro wound healing assay a standard in vitro technique for probing collective cell migration was used according to the recommendations (Jonkman et al., 2014 (link)).
We compared the dynamics of the migration into the cell-free area of 2D eMSCs, treated with culture medium, containing 1% FBS and 10 ng recombinant human VEGF (Gibco Life Technologies, United States) or conditioned growth medium from 2D and 3D eMSCs, containing 1% FBS. Two-well silicone insert (Ibidi) in a 24-well plates were used for gap creation. Twenty-five thousand cells in growth medium were seeded on both sides of the insert and incubated overnight after a confluent monolayer was formed. The next day inserts were removed, cells were washed and compared samples of medium were added to the wells. Photographs of the cells were taken at 0, 8 and 24 h to monitor the gap close. The gap size was measured as a function of time using Fiji/ImageJ software package.
We compared the dynamics of the migration into the cell-free area of 2D eMSCs, treated with culture medium, containing 1% FBS and 10 ng recombinant human VEGF (Gibco Life Technologies, United States) or conditioned growth medium from 2D and 3D eMSCs, containing 1% FBS. Two-well silicone insert (Ibidi) in a 24-well plates were used for gap creation. Twenty-five thousand cells in growth medium were seeded on both sides of the insert and incubated overnight after a confluent monolayer was formed. The next day inserts were removed, cells were washed and compared samples of medium were added to the wells. Photographs of the cells were taken at 0, 8 and 24 h to monitor the gap close. The gap size was measured as a function of time using Fiji/ImageJ software package.
5
Isolation and Culture of BMECs
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The BMECs were cultured based on the previously published instructions. 33 (link) The subchondral area of the femoral head was used to extract the cancellous bone. Digestible bone fragments were treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) and 0.2% type I collagenase for 5 min. The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, USA). The lytic products were filtered using a 70-μmol/L cell filter and centrifuged for 6 min at 1500 rpm. The supernatant was removed, and the cell sediments were exposed to an endothelial cell medium (ECM) (ScienCell Research Laboratories, Carlsbad, USA) containing 100 U/mL penicillin, 100 U/mL streptomycin, 5 mL recombinant human VEGF, and 5% fetal bovine serum (FBS) (Gibco) in 5% CO 2 in a 37°C wet incubator. For cell passage, the medium was removed. The cells were washed using phosphate-buffered saline (PBS) and treated with 0.25% trypsin-EDTA for 5 min. The reaction was ceased using a medium with 10% FBS. The cells were then repeatedly blown using a 1-microliter pipette tip. Cell passage was performed when cell confluence achieved 90%, and the 3 rd cell generation was used for the following procedures.
6
Hydrogel Synthesis and Biomolecule Functionalization
7
Evaluation of Anti-Angiogenic Drug Effects
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Sunitinib and sorafenib were from LC Laboratories (Woburn, MA, USA). Sodium bicarbonate and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were from Sigma-Aldrich. Recombinant human VEGF was from Peprotech (#100-20-10). Cycloheximide was purchased from Santa Cruz biotechnology (#SC-3508). For Western blot analysis, the following primary antibodies and concentrations were used: anti-phospho-Akt antibody (1:500) (#4060; Cell Signaling Technology), anti-Akt antibody (1:1000) (#2920; Cell Signaling Technology), anti-VEGFR-2 antibody (1:1000) (#2479; Cell Signaling Technology), anti-VEGFR-1 (1:500) (#sc-316; Santa Cruz biotechnology), anti-β-actin antibody (1:5000) (#A2228; Sigma Aldrich), anti-Tie-2 antibody (1:500) (#MAB313; R&D systems) and anti-FGFR-1 antibody (1:1000) (#sc-121; Santa Cruz Biotechnology). Immunohistochemical staining were performed with anti-CD31 antibody (#Rb-10333-PO; Thermo Scientific). For immunofluorescence anti-CD31 (1:50) (# 553370; BD Pharmigen) and anti-VEGFR-2 antibody (1:50) (#2479; Cell Signaling Technology) were used. For flow cytometry, the following antibodies and dilutions were used: anti-CD31 (1:100) (#17-0319; eBioscience), anti-avb3 integrin (1:100) (MAB1976; Millipore), anti-VCAM-1(1:100) (#12-1069; eBioscience), anti-ICAM-1 (1:100) (#12-0549; eBioscience) and anti-MCP-1 (1:100) (#12-7099; eBioscience).
8
Angiogenesis Assay with Arnebin-1 and VEGF
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Arnebin-1 was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and recombinant human VEGF was from PeproTech Inc. (Rocky Hill, NJ, USA). Growth factor-reduced Matrigel basement membrane matrix was obtained from BD Biosciences (Bedford, MA, USA). Medium 199 (M199) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LY294002, a PI3K inhibitor, was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents utilized were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
9
Trastuzumab, VEGF, and Squalamine Protocol
10
VEGF and GDNF Encapsulated PLGA Nanoparticles
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Recombinant human VEGF (PeproTech, London, UK) and recombinant murine GDNF (Peprotech) enclosing PLGA (50:50; lactic/glycolic [%]) (Resomer® RG 503; Boehringer Ingelheim, Ingelheim, Germany) NS were prepared as previously described.20 (link)
Briefly, 133 mg PLGA 50:50 (previously sterilized by gamma radiation) were dissolved in 3.33 mL dichloromethane and emulsified with 200 μL of 0.15% w/v Recombinant human VEGF or recombinant murine GDNF aqueous solution (containing 7% [w/v] human serum albumin and 2.5% [w/v] poly-ethylene-glycol 400) by probe sonication for 30 seconds at 50 W (Branson® Sonifier® 250, Biogen, Derio, Spain). The first emulsion was poured into 5% (w/v) polyvinyl alcohol solution and sonicated again for 1 minute, in order to obtain a double emulsion (w1/o/w2). This emulsion was poured into 2% (v/v) isopropanol solution and stirred at room temperature for 2 hours to promote the removal of the organic solvent. The newly formed NS were separated by centrifugation at 20,000× g, resuspended in 2.5% (w/w in respect to PLGA) trehalose aqueous solution and freeze-dried for 24 hours. The entire process of NS preparation was conducted under aseptic conditions. Empty NS (without VEGF or GDNF) were prepared using the same method described above.
Briefly, 133 mg PLGA 50:50 (previously sterilized by gamma radiation) were dissolved in 3.33 mL dichloromethane and emulsified with 200 μL of 0.15% w/v Recombinant human VEGF or recombinant murine GDNF aqueous solution (containing 7% [w/v] human serum albumin and 2.5% [w/v] poly-ethylene-glycol 400) by probe sonication for 30 seconds at 50 W (Branson® Sonifier® 250, Biogen, Derio, Spain). The first emulsion was poured into 5% (w/v) polyvinyl alcohol solution and sonicated again for 1 minute, in order to obtain a double emulsion (w1/o/w2). This emulsion was poured into 2% (v/v) isopropanol solution and stirred at room temperature for 2 hours to promote the removal of the organic solvent. The newly formed NS were separated by centrifugation at 20,000× g, resuspended in 2.5% (w/w in respect to PLGA) trehalose aqueous solution and freeze-dried for 24 hours. The entire process of NS preparation was conducted under aseptic conditions. Empty NS (without VEGF or GDNF) were prepared using the same method described above.
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