Fifty micrograms of total cell lysate was separated on a 10% SDS-PAGE gel, transferred on PVDF membrane and blotted with antibodies. HGF, VEGFA, VEGFR2 and MMP2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ERK1/2, phospho-ERK1/2 and α-Tubulin were from Cell Signaling and Sigma, respectively. HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were used to identify the protein of interest. Each experiment was repeated at least two times. Secreted VEGFA and HGF was detected either in conditioned media (CM) of cultured cells or patient's sera using ELISA kits from Ray Biotech (Norcross, GA, USA).
Vegf a
Manufactured by RayBiotech
Sourced in United States
VEGF-A is a protein that stimulates the growth of blood vessels. It plays a key role in the formation of new blood vessels, a process called angiogenesis. VEGF-A is an important regulator of both physiological and pathological angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hepatocyte growth factor (hgf), by RayBiotech (2 mentions)
Basic fibroblast growth factor (bfgf), by RayBiotech (2 mentions)
Interleukin 6 (il 6), by RayBiotech (2 mentions)
Ang 2, by RayBiotech (2 mentions)
Mmp 2, by Santa Cruz Biotechnology (1 mentions)
Hepatocyte growth factor (hgf), by Santa Cruz Biotechnology (1 mentions)
Vegfa, by Santa Cruz Biotechnology (1 mentions)
Vegfr2, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Vegfr2, by RayBiotech (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Vegf a, by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by Abcam (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Automated cell counter, by Beckman Coulter (1 mentions)
Tgf β, by MyBioSource (1 mentions)
9 protocols using vegf a
1
Quantification of Angiogenic Factors
2
Quantification of Angiogenic Factors
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Enzyme-linked immunosorbent assay (ELISA) was used to quantify urinary and serum levels of NRP-1 (Cloud-Cone Corp, Katy, TX, USA), VEGFA (RayBiotech, Norcross, GA, USA), VEGFRI (RayBiotech, Norcross, GA, USA), VEGFR2 (RayBiotech, Norcross, GA, USA), and SEMA3A (Cloud-Cone Corp, Katy, TX, USA) following the manufacturer’s instructions. Triplicate for all samples were done. Urinary levels were normalized against the corresponding urine creatinine levels.
3
Quantifying Secreted Angiogenic Factors
4
Bioactive Factors Secreted by SVF Cells
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Fresh mSVF pellet was suspended in minimum essential medium (MEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin. Conditioned medium was collected from SVF cells and after two days of single culture without media change and was analyzed immediately by following ELISA kits: basic fibroblast growth factor (bFGF; RayBiotech, Peachtree Corners, GA, USA; #ELR-bFGF-1; detection range 2–500 pg/mL), vascular endothelial growth factor A (VEGF-A; RayBiotech; Peachtree Corners, GA, USA; #ELR-vEGF-1; detection range 2–200 pg/mL) and tumor growth factor beta (TGF-b; Invitrogen, Carlsbad, CA, USA; TGF beta-1 Rat ELISA Kit; detection range: 31.25–2000 pg/mL).
5
Plasma Biomarker Profiling Protocol
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Plasma samples were obtained by venipuncture after an overnight fasting. Glucose was analyzed by an automated analyzer (Hitachi Modular P800, Roche, Basel, Switzerland). Serum concentrations of triglycerides and free fatty acids (FFA) were determined by using commercially available kits (Infinity, Thermo Electron Corporation, Melbourne, Australia). The carcinoembryonic antigen (CEA), fibrinogen and high sensitivity C-reactive protein (CRP) concentrations were measured as previously reported [11 (link)]. White blood cell (WBC) count was determined using an automated cell counter (Beckman Coulter, Inc., Fullerton, CA, USA). Circulating levels of MAGP-1, IL-4, IL-6, IL-13, VEGFA, OPN (RayBiotech, Inc., Norcross, GA, USA) and TGF-β (Mybiosource, San Diego, CA, USA) were assessed by commercially available ELISA kits according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were <10 and <12% for all analysed molecules.
6
Serum Biomarker Quantification by ELISA
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7
Quantification of Secreted Neurotrophic and Vasculogenic Factors
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Secreted neurotrophic and vasculogenic proteins were determined by ELISA. Stem cells
were seeded at 6500 cells/cm2 per 5 mL media in 25 cm2 flasks. The
media from the cells was changed after 4 days and replaced with fresh 5 ml media, which
was then collected and analyzed at day 7 with BDNF, VEGF-A and angiopoietin-1 sandwich
ELISA kits according to the manufacturer’s protocols (RayBiotech, GA, USA). All samples
were analysed in duplicate and the end-absorbance was measured at 450 nm (BioTek Synergy
microplate reader). Quantity of secreted protein was calculated in
pg/ml/104cells which was determined from data obtained from protein standard
curves and final cell counts after 7 days of culture.
were seeded at 6500 cells/cm2 per 5 mL media in 25 cm2 flasks. The
media from the cells was changed after 4 days and replaced with fresh 5 ml media, which
was then collected and analyzed at day 7 with BDNF, VEGF-A and angiopoietin-1 sandwich
ELISA kits according to the manufacturer’s protocols (RayBiotech, GA, USA). All samples
were analysed in duplicate and the end-absorbance was measured at 450 nm (BioTek Synergy
microplate reader). Quantity of secreted protein was calculated in
pg/ml/104cells which was determined from data obtained from protein standard
curves and final cell counts after 7 days of culture.
8
Profiling Angiogenic Factors in EC/VSMC
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Saphenous vein-derived ECs and VSMCs were separately cultured on 6-well plates and treated with TGFβ1 (0, 1, and 10 ng/ml) for 24 h. The resulting conditioned medium was harvested and used to determine the relative levels of AGFs by ELISA (VEGF-C, Ang-1, TGF-β1; R&D Systems Ltd) or Quantibody multiplex array (Angiogenin, Ang-2, EGF, bFGF, HB-EGF, HGF, Leptin, PDGF-BB, PlGF, VEGF-A; Raybiotech, Norcross, United States) according to the manufacturers’ instructions. Each experiment was performed on three separate occasions.
9
Hypoxia and Stress Signaling Pathways in Cultured Cells
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Isolated cells were cultured in DMEM (Dulbecco's Modi ed Eagle Medium, D5648Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum/FBS (Invitrogen, Rockford, IL, USA), 1% penicillinstreptomycin (10,000 U/ml), [Cat no-15140122] from Invitrogen at 37 °C with 5% CO 2 in a humidi ed incubator. Thea avin 3, 3'digallate/TF3 [Cat no-92223-1MG] and Methotrexate/MTX [Cat no-M8407] were purchased from (Sigma, St Louis, MO, USA). For protein estimation, Bradford reagents were obtained from bio rad (Cat no-5000006). Primary antibodies for human [HIF-1α(ab51608),GRP78(#3183), CHOP(#2895), HSP-70(#4876), Bcl-2(#4223), Bax (#2772), Beclin-1(#3738) p62 (#5114), α-tubulin (#2144), 10x cell lysis buffer Cat no-#9803 and Protease Inhibitor Cocktail, PKC (100x) Cat no-5871] were obtained from Cell-Signaling Technologies Inc. (Danvers, MA, USA). Nitrocellulose membrane was procured from MF-Millipore [Cat no-HAWP04700] 0.45 µm pore size. Caspase-9 and Caspase-3 were obtained from Biolegend (Cat No: 621902 and 634101 respectively). Anti-LC3b antibody (ab51520) was procured from Abcam. The concentration of VEGF-A and ANG-1 ELISA Kits [(ELH-VEGF-1) and (IQH-Angiopoietin1-1)] were procured from (Ray Biotech, Norcross, GA). For detection of apoptosis FITC Annexin V Apoptosis Detection Kit, I (Cat no-556547) was used from BD, Biosciences.
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