Wizard sv lysis buffer

The concentration of VEGF in the lysates of HUVECs was measured using a Human VEGF-A ELISA kit (RayBio, Peachtree Corners, GA, USA) following the manufacturer’s protocol. Initially, HUVECs were treated with BCP (10 and 20 μM) or 0.5% DMSO (as solvent control) for 6 h. Wizard^® SV Lysis buffer (Promega, Madison, WI, USA) was used for preparation of cell lysates. The standards and the lysates of HUVECs were then pipetted into the wells (in triplicate) of the 96-well plate of the kit, which were coated with human VEGF specific antibody. The plate was incubated at 15 °C for 24 h to facilitate the efficient VEGF binding with the immobilized antibody. Then, following the washing of the cells with a wash buffer solution, anti-human VEGF biotinylated antibody was added to each well. The wells were subsequently washed followed by addition of HRP-conjugated streptavidin. Afterwards, addition of tetramethylbenzidine substrate solution resulted in blue color formation, with the intensity of color being proportional to VEGF content. Then, sulphuric acid (stop solution) was added to the wells, turning blue color to yellow. Lastly, the absorbance was measured using a microplate reader at 450 nm. A regression equation was used for the estimation of VEGF concentration (pg/mL of cell lysates) and the results were presented as the mean ± SD.

The treated MCF-7 cells were harvested and lysed by using Wizard® SV lysis buffer (Promega, Madison, USA). The assay was run by using the ELISA kit of human VEGF-165 according to the manufacturers' protocol (Raybio, USA).