Human angiogenesis antibody array

BMSCs and aBMSCs were washed with serum-free MEM α, followed by the addition of 5 ml serum-free MEM α before the cells were incubated at 37°C for 24 hours. After incubation the cells and conditioned media were harvested. Relative levels of 55 angiogenesis related proteins were detected in this conditioned media using the Human Angiogenesis Antibody Array (R&D Systems, Minneapolis, MN; Cat. #ARY007). The DNA concentration was measured from the harvested cells. Array membranes were analyzed by quantifying the mean spot pixel densities using the freeware Image J software (NIH http://rsb.info.nih.gov/ij). Samples were standardized using DNA content.

DPSC cells were grown in either 15% FBS or 15% Human Sera containing alpha minimum essential medium (αMEM). At passage 11(DPSC-FBS) and 12(DPSC-HS), cell layers were washed with serum free αMEM, followed by the addition 5 ml of serum free αMEM before the cells were incubated at 37°C for 24 hours. After incubation the cells and conditioned media were harvested. Relative levels of 55 angiogenesis related proteins were detected in this conditioned media using the Human Angiogenesis Antibody Array (R&D Systems; Cat. #ARY007). The DNA concentration was measured from the harvested cells. Array membranes were exposed to xray film. These films were analyzed by quantifying the mean spot pixel densities using the freeware Image J software (NIH (http://rsb.info.nih.gov/ij)). Samples were standardized using DNA concentrations.

The secretome obtained from the transfected (secretome^146a) and nontransfected hASCs (secretome) was assessed using a human angiogenesis antibody array and human cytokine array (R&D Systems). The two arrays were used to detect the differences in composition between the two different types of the secretome by analyzing the relative levels of 55 angiogenic related proteins and 36 different cytokines, chemokines, and acute-phase proteins. The studies were carried out according to the manufacturer’s protocols. Images of the corresponding chemiluminescent dot blots were taken, and the mean spot pixel density for each protein was calculated using the ImageJ software.

Secreted angiogenesis mediators were identified in fallopian tube secretory cell and ovarian carcinoma cell conditioned media using the Human Angiogenesis Antibody Array (R&D systems: ARY007). ELISAs was employed for the precise quantification of VEGF (R&D systems: DVE00) and SERPINE1 (R&D systems: DSE100) from conditioned media as recommended by the manufacturer’s protocol. Fresh DMEM/F12 or RMPI media were tested and used as a negative control.