Retinas were homogenized with a bullet blender 24 Gold (Next Advance, NY, USA) in T-PER buffer (78510, ThermoFisher Scientific, Scoresby, Victoria, Australia) containing 5 mM EDTA (AM9260G, ThermoFisher Scientific, Scoresby, Victoria, Australia) and protease/phosphatase inhibitor cocktail (1:100, Sigma-Aldrich, St. Louis, MO, USA). Total protein concentration was quantified via colorimetric assay (5000207 & 5000205 Bio-Rad Laboratories, CA, USA). VEGF was measured in retinal lysates using a commercially available ELISA kit (DY493, R&D Systems). Five to eight mice were analyzed in duplicate per group.
Dy493
Manufactured by R&D Systems
Sourced in Germany
DY493 is a protein purification column designed for the separation and purification of proteins from complex biological samples. The column utilizes a proprietary resin to facilitate efficient protein capture, separation, and recovery. The core function of the DY493 is to provide a reliable and reproducible method for the purification of target proteins from cell lysates, tissue homogenates, or other complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Am9260g, by Thermo Fisher Scientific (1 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Bullet blender 24 gold, by Next Advance (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Dy406, by R&D Systems (1 mentions)
Dy410, by R&D Systems (1 mentions)
Dy479, by R&D Systems (1 mentions)
4 protocols using dy493
1
Quantifying VEGF in Retinal Lysates
2
Quantifying VEGF in Cell Media
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For quantification of free VEGF present in the media, ECs were plated at a density of 2.5×105 cells per well of a 6-well plate. Cells were chilled to 4°C, medium was removed and cells were washed twice in ice-cold PBS. Medium was replaced with ice-cold Opti-MEM containing VEGF at a final concentration of 30 ng/ml. Media were diluted 1:10 in 1% BSA in PBS, and VEGF concentration was quantified using a mouse VEGF duo-set ELISA, according to the manufacturer's instructions (DY493, R&D Systems).
3
Macrophage Cytokine Secretion Assay
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Macrophage colony‐stimulating factor differentiated bone marrow‐derived macrophages were plated in 1500 μL/well in 10% FCS, 1% PS in RPMI. Medium was either supplemented with 10 or 50 ng/mL lipopolysaccharide (Sigma, K235). Cells were cultured for 24 hours and thereafter, medium was collected for ELISA as well as aortic ring assay, after which Trizol was added for RNA isolation. Supernatant was stored at −80 °C. CCL2 (C‐C motif chemokine ligand 2), IL‐6 (interleukin‐6), TNF‐α (tumor necrosis factor alpha), and VEGF concentrations were determined by ELISA according to manufacturer's protocol (R&D systems, DY406, DY410, DY479, and DY493).
4
ELISA Quantification of Serotonin, Hmgb1, and Vegf
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