CellTiter 96® Aqueous One Solution Cell Proliferation assay (MTS) and Caspase-Glo 3/7 Assay Kit were both obtained from Promega Corporation (Madison, WI, USA). Hoechst 33342 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dead Cell Apoptosis Kit with Annexin V Alexa Fluor® 488 and propidium iodide (PI), and fetal bovine serum (FBS) were from Invitrogen (Grand Island, NY, USA). Quantikine Human VEGF Immunoassay Kit was purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies for Bad (C-7) (#8044), Bcl-xL (H-5) (#8392), and GAPDH (0411) (#47724) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Bcl-2 (#2872S), HIF-1α (#14179S), hVEGF (#8065), FasL (#4273S), FADD (#2782S), DR5 (#3696S), monoclonal pro-caspase-9 (C9) (#9508S), pro-caspase-3 (#9662S), and pro-caspase-7 (#9492S) antibodies and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody HIF-1β (#611079) was from BD Biosciences (San Jose, CA, USA). The BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA). HaltTM Protease and Phosphatase Inhibitor Single-Use Cocktail were purchased from Life Technologies (Grand Island, NY, USA). EDTA solution was form Thermo Scientific (Rockford, IL, USA).
Quantikine human vegf immunoassay kit
Manufactured by R&D Systems
Sourced in United States
The Quantikine Human VEGF Immunoassay kit is a quantitative sandwich enzyme immunoassay designed for the measurement of human vascular endothelial growth factor (VEGF) concentrations in cell culture supernates, serum, and plasma.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh 0411, by Santa Cruz Biotechnology (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Edta solution, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay mts, by Promega (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
M199 medium, by Welgene (1 mentions)
Type 1 collagen, by BD (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Et 1 elisa kit, by R&D Systems (1 mentions)
15 protocols using quantikine human vegf immunoassay kit
1
Cell Proliferation and Apoptosis Assay
2
Quantifying VEGF Secretion in Cancer Cell Lines
3
VEGF Secretion Inhibition by CHSP
4
Quantifying VEGF Secretion Inhibition
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The effect of GA on VEGF protein secretion was analyzed by ELISA with a Quantikine Human VEGF Immunoassay kit (R&D Systems, Minneapolis, MN, USA), targeting VEGF165 in the cell culture supernatant. Cells (6×105) were seeded in 60-mm cell culture dishes and allowed to attach to the substrate and grow for 16 h at 37°C before treatment with various concentrations of GA for 24 h. Culture supernatants were collected for the VEGF assay. The inhibition of VEGF protein secretion was expressed as a percentage of the control. Cell lysates were also assayed for total protein levels using the BCA protein assay kit (Pierce, Rockford, IL, USA) to adjust VEGF levels.
5
VEGF Secretion Inhibition Assay
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The effects of the nine compounds on VEGF protein secretion were analyzed by performing an ELISA with a Quantikine Human VEGF immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA), targeting VEGF165 in the cell culture supernatant. Cells (6×105) from the two cell lines were seeded in 60-mm cell culture dishes and allowed to attach to the substrate and grow for 16 h prior to treatment with 40 μM of each compound or without, which served as a control, for an additional 24 h. The culture supernatants were collected for the VEGF assay and the inhibition of VEGF protein secretion was expressed as a percentage of the control.
6
Nobiletin Modulates VEGF Secretion
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Secreted vascular endothelial growth factor (VEGF) protein levels were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) with a Quantikine Human VEGF Immunoassay Kit from R&D Systems (Minneapolis, MN) targeting VEGF in cell culture supernates. Cells (104/well) were seeded into 96-well plates and incubated for 16 h before being treated with 0 to 160 μg/ml nobiletin in triplicates for 24 h with DMSO as solvent control. Culture supernates were collected for VEGF assay. VEGF levels were determined following the manufacturer's instructions. A total of 3 independent experiments, each in triplicates, were assayed, and the mean VEGF protein level from each duplicate was used for statistical analysis.
7
Quantitative VEGF Assay Protocol
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Culture medium was assayed for VEGF concentration using the Quantikine® Human VEGF Immunoassay Kit (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Cell culture medium was collected and supplemented with 2% fetal bovine serum (Hyclone) before storage at -20°C for maintaining the stability of VEGF. All reagents and standards were freshly prepared and added during the assay as instructed by the manufacturer. The concentration of VEGF was measured by the color intensity of solutions using a microplate reader (Bio-Tek) at 450 nm and 570 nm, respectively. Readings at 570 nm were subtracted from the readings at 450 nm to allow correction for optical imperfections. VEGF concentrations were obtained by comparing the corresponding readings with those of the standard curve using known concentrations of VEGF. Assays were repeated in duplicate.
8
Isolation and Characterization of Gintonin
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Crude gintonin was isolated from P. ginseng as described previously [19] . Gintonin is a glycolipoprotein containing ginseng protein complexed with LPA [20] (link). Ginsenosides were purchased from the LKT Laboratories Inc. (St. Paul, MN, USA). VEGF, basic fibroblast growth factor, and Quantikine human VEGF immunoassay kit were purchased from R&D Systems (Minneapolis, MN, USA). M199 medium and 0.1% gelatin solution were purchased from WelGENE (Daegu-si, Korea). Matrigel (growth factor reduced) and collagen type 1 were purchased from BD Biosciences (Bedford, MA, USA). All other reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Quantification of VEGF Protein Levels
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The levels of VEGF in cell culture supernatants were analyzed by a Quantikine Human VEGF Immunoassay kit (R&D Systems, Minneapolis, MN, USA). Cells (1×104/well) were seeded into 96-well plates and incubated overnight. Subsequently, the cells were treated with nobiletin for 16 h in serum-free medium. Culture supernatants were collected and spun down at 10,000 g at 4°C. The supernatant was collected and stored at −70°C. The amounts of VEGF were measured following the manufacturer’s instructions, and normalized to cell numbers for each treatment. A total of three independent experiments, each in triplicates, were assayed, and the mean VEGF protein level from each triplicate was used for statistical analysis.
10
Quantification of ET-1 and VEGF Release
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The release of ET-1 and VEGF in the conditioned media of serum-starved cells was measured on microtiter plates by ET-1 ELISA kit and by Quantikine human VEGF immunoassay kit (R&D Systems, MN, USA) according to the manufacturer's instructions.
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