Various concentrations of anti‐VEGFR2 scFvs were mixed with 3 nM human VEGF‐A (Peprotech), and the mixture was added to 96‐well plates coated with 1 μg/mL VEGFR2‐Fc and preblocked with 1% BSA. After incubation for 1 hour at room temperature and washing with PBST, the bound VEGF molecules were detected using anti‐VEGF mAb (GeneTex) and HRP‐labeled goat anti‐mouse IgG. Detection was accomplished with a mixture of o‐phenylenediamine dihydrochloride and H2O2, and the reaction was terminated with 3 N HCl. The absorbance was determined using a microplate reader at 490 nm.
Human vegf a
Manufactured by Thermo Fisher Scientific
Sourced in Sweden
Human VEGF-A is a recombinant protein that corresponds to the human vascular endothelial growth factor A (VEGF-A) sequence. VEGF-A is a key regulator of angiogenesis, the process of new blood vessel formation, and plays a critical role in various physiological and pathological processes.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Cellrox, by Thermo Fisher Scientific (1 mentions)
H2dffda, by Thermo Fisher Scientific (1 mentions)
Plgf 1, by Thermo Fisher Scientific (1 mentions)
2 nbdg, by Merck Group (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Methylglyoxal, by Merck Group (1 mentions)
Forskolin, by R&D Systems (1 mentions)
Vegf a, by Thermo Fisher Scientific (1 mentions)
Microbeads kit, by Miltenyi Biotec (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Y27632, by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Stempro 34 sfm, by Thermo Fisher Scientific (1 mentions)
4 protocols using human vegf a
1
VEGF-A Binding Inhibition Assay
2
Optimizing Cell Culture for VEGF/PlGF Research
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Cell culture media RPMI 1640 Medium GlutaMAXTM was obtained from Life Technologies. Human VEGF-A and PlGF-1 were from Peprotech. H2DFFDA and CellROX were from Life Technologies. Hydrogen peroxide, methylglyoxal, NAC and 2-NBDG were from Sigma Aldrich. All the primers for qPCR were custom synthesised from Sigma Aldrich. Primer sequences are described in Supplementary Table S3 .
3
Immunofluorescence Assay for VEGF-A Stimulation
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1 × 105 transduced HDMECs were plated on coverslips in six well plates. Confluent cells were starved overnight in DMEM supplemented with 1% BSA. Starved cells were gently washed twice with PBS, and stimulated with 50 ng/ml human VEGF-A (Peprotech, Sweden) for 10 min at 37°C. VEGF-A containing medium was quickly removed and cells were washed twice in ice-cold PBS. Subsequently, cells were fixed in 4% paraformaldehyde solution at room temperature for 15 min, and blocked with 2% BSA in PBS for 1 hr without permeabilization. Further incubation steps with primary and secondary antibodies were described as below.
4
Directed Differentiation of iPSCs to iPS-EC
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The method used was adapted from previously published protocols23 (link). iPS cells were seeded on Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (SLS, 356231) at a density of 1.6 × 105 cells per 6 well in StemMACS iPS-Brew XF (Milteney Biotech, 130-104-368) supplemented with 10 μM Y-27632 (ATCC ACS-3030) for 24 h. The following day (day 1), the medium was changed to N2B27 medium, a 1:1 ratio of Neurobasal medium (Thermo, 21103049) and DMEM/F12 (Gibco, 11330-032), with N2 (Thermo, 17502048), B27 (Thermo, 12587010), Glutamax (Thermo, 350050061) and freshly supplemented with 8 μM of CHIR (Sigma, SML1046) and 25 ng/ml of BMP4 (ThermoFisher, PHC9534). After 72 h (day 4), the medium was replaced with StemPro-34 SFM (Gibco, 10639011) supplemented with 200 ng/ml human VEGFA (Peprotech, 100-20) and 2 μM Forskolin (R&D system, 1099). On day 6, cells were selected by Magnetic Activated Cell Sorting (MACS) for iPS-EC expressing CD144 using Microbeads Kit (Miltenyi Biotec, 130-097-857). Positive cells were seeded on mouse Collagen IV (BioTechne, 3410-010-02) coated plate in EGM2-MV medium (Promocell, C-22011) supplemented with 20% FBS (Thermo, 10500064) and 50 ng/ml VEGFA. Cells were used for experiments up to 3 passages.
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