A human VEGF immunoassay kit by Quantikine (R&D Systems, Minneapolis, MN, USA) was used for the determination of VEGF levels in serum and tears. The quantitative sandwich enzyme immunoassay technique was used according to the manufacturer’s instructions. For the initial step, 100 μL of RDW1 assay solution was placed onto the microplate. Then, 100 μL of the sample (serum and tear fluid) was added to the microplate and incubated for 2 hours at room temperature. Next, 200 μL of conjugate was added after three washings, followed by re-incubation at room temperature. Subsequently, it was subjected to three more washings before being centrifuged at 25 rpm, together with 200 μL of substrate, and incubated. The last solution that was added was 50 μL of stop solution. Finally, the plates were read using a microplate reader at 450–570 nm wavelength.
Human vegf immunoassay kit
Manufactured by R&D Systems
Sourced in United States
The Human VEGF Immunoassay Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of VEGF (Vascular Endothelial Growth Factor) levels in human cell culture supernates, cell lysates, serum, and plasma samples. The kit utilizes a VEGF-specific antibody coated on a microplate to capture VEGF in the sample. A second VEGF-specific antibody conjugated to horseradish peroxidase is then used for detection and quantification of the captured VEGF.
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6 protocols using human vegf immunoassay kit
1
Quantitative VEGF Immunoassay in Serum and Tears
2
Quantifying VEGF Secretion from NCI-H460 Cells
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The quantity of VEGF released from NCI-H460 cells into the culture medium was determined using a human VEGF immunoassay kit (R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s instructions. Briefly, the culture medium was cleared via centrifugation at 4,000 g for 10 min at 4°C. The recombinant VEGF standards and cleared medium samples were loaded into a 96-well immunoplate pre-coated with a VEGF monoclonal antibody. The samples were incubated for 1 h at room temperature (RT). After 3 washes, a horseradish peroxidase (HRP)-conjugated polyclonal antibody against VEGF was added to each well and incubated for 3 h at RT. After 3 washes, color development was initiated by adding a substrate solution and was monitored at 450 nm using a microplate reader (Emax, Molecular Devices). Extracellular VEGF was quantified based on the standard curve.
3
Quantifying VEGF Protein in EPC Cultures
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Enzyme-linked immunosorbent assays (ELISAs) were performed to quantify the VEGF protein in the medium after transfection36 (link). The culture supernatants that were harvested from EPCs at different passages 2 h, 24 h and 48 h after UMT were collected and stored at − 80 °C. The amount of VEGF in each supernatant was quantified by a human VEGF immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the recommended procedures. The optical density was measured by a SpectraMAX 190 absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450–540 nm.
4
Quantifying VEGF in Tear Samples
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First, as a point of reference for VEGF, total protein concentrations were determined in tear samples with the BCA Microplate method (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Human albumin was used as standard.
For the quantitative determination of VEGF in tear fluid we used a human VEGF immunoassay kit by Quantikine (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. This assay employs the quantitative sandwich enzyme immunoassay technique.
For the quantitative determination of VEGF in tear fluid we used a human VEGF immunoassay kit by Quantikine (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. This assay employs the quantitative sandwich enzyme immunoassay technique.
5
Quantifying Angiogenic Factors in Cell Co-Culture
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In order to measure ANG-1, ANG-2 and VEGF protein levels in cell co-culture media, samples were collected, centrifuged and processed immediately. For the determination of VEGF concentration in the HUVEC/MCF-7 cell co-culture media a human VEGF Immunoassay kit (R&d Systems Europe Ltd.) was used. The samples (in triplicate) were processed according to the supplier's instructions. At the end of the procedure, absorbance was determined at a wavelength of 450 nm, with corrections at 540 nm. For the determination of ANG-1 and ANG-2 concentration in the HUVEC/MCF-7 cell co-culture media we used a Human Angiopoietin-1 or -2 Immunoassay kit (R&d Systems Europe Ltd.) following the supplier's instructions.
Statistical analysis. data are expressed as the mean ± standard errors of the mean (SEM). Statistical differences between groups were analyzed using one way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. Results were considered as statistically significant at P<0.05.
Statistical analysis. data are expressed as the mean ± standard errors of the mean (SEM). Statistical differences between groups were analyzed using one way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test. Results were considered as statistically significant at P<0.05.
6
VEGF Quantification in SH-SY5Y and Co-Culture Cells
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For the determination of the concentration of VEGF in the SH-SY5Y cell cultures and the HuVEC/SH-SY5Y cell co-culture media, a Human VEGF Immunoassay kit (R&d Systems Europe Ltd.) was used. The samples (in triplicate) were processed according to the manufacturer's instructions. Briefly, SH-SY5Y cells were seeded into 96-multiwell plates at a density of 8x10 3 cells/well in DMEM/Ham's Nutrient Mixture F-12 supplemented with 10% FBS and incubated at 37̊C for 24 h. Next, the media were aspirated and replaced by fresh media supplemented with 0.5% sFBS and containing either 1 mM or 1 nM of melatonin or vehicle (ethanol). After 24 h of incubation, the media were collected, centrifuged to remove particles and subjected to ELISA according to the manufacturer's instructions. For the determination of the protein levels of VEGF in the cell co-culture media, samples were collected, centrifuged and immediately processed. At the end of the procedure, the absorbance was determined at a wavelength of 450 nm, with the correction wavelength set at 570 nm.
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