Human vegf a elisa kit

CAFs were transfected with miR-101-3p mimics, miR-NC, miR-101-3p-inhibitor or inhibitor NC for 24 h. The supernatants were collected to detect VEGFA using an human VEGFA ELISA Kit (RayBiotech Life, GA) following the manufacturer’s instructions.

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.

HUVEC and MDA-MB-231 cells were seeded on six-well plates at 1 × 10⁵ cells per well on a 96-well plate. After overnight culture, the cells were exposed to C3 for three days. The expression of VEGF in these cells was compared in normoxic and hypoxic conditions. Following treatment, the supernatants were collected and centrifuged to pellet any debris. The cells were then lysed with a RIPA Lysis and Extraction Buffer (Pierce Biotechnology, Rockford, IL, USA) and the total protein was measured using Bio-Rad protein assay (Bio-Rad Laboratories). VEGF production was measured using the human VEGF-A ELISA Kit (RayBiotech, Norcross, GA, USA). The experiment was performed in duplicate, and the results are shown as mean ± SD.

VEGFA levels in supernatants were measured by ELISA as previously described 8 (link). Briefly, the supernatants were collected, and VEGFA levels were measured by human VEGFA ELISA kit in accordance with the kit instruction (RayBiotech Life, Peachtree Corners, GA). The absorbance at A450 was measured on a microplate reader (BioTek, Santa Clara, CA).