Human angiogenesis antibody array

In order to overcome the possibility that a different number of cells alter the results of chemokines analysis, growth curves for each CRC cell line were assessed and cells were seeded at different cell concentrations to evaluate their rate growth. For cell counting, Thoma chamber was used.
Standardization: all cell lines were plated into 60 × 15 dishes (BD Falcon, Oxford, UK) to have about 1×10⁶ cells after 48 h of plating. After 24 h from the plating, the culture medium was replaced by serum-free medium, and after 24 h, media were collected and cells were counted.
Assessment: CRC cell culture media were analyzed in triplicate as per the manufacturer’s instructions using human IL-8 (Enzo Life Sciences, Farmingdale, NY, USA) and VEGF (R&DSystems, MN, USA) specific ELISA. Absorbance was read at 450 nm. IL-8 and VEGF expression was represented as pg/mL and then related to the control. HCT116 Parental and HCT116 PTEN^−/− culture media were analyzed by Human Angiogenesis Antibody Array (RayBiotech, Norcross, GA, USA), according to the manufacturer’s protocol.

MDA-MB-231 (Caucasian American) and MDA-MB-468 (African American) TNBC cells were purchased from the American Type Culture Collection (ATCC). Heat-inactivated fetal bovine serum (HI-FBS), Dulbecco’s modified Eagle’s medium (DMEM) high glucose, penicillin/streptomycin, and phosphate-buffered saline (PBS) were obtained from Genesee Scientific (San Diego, CA, USA). Alamar Blue^®, fucoxanthin (95% purity), dimethyl sulfoxide (DMSO), chloroform, and isopropyl alcohol were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Human Angiogenesis Antibody Array (Cat# AAH-ANG-1000-4) and TNF-alpha (Cat# ELH-TNFα) were purchased from RayBiotech (Norcross, GA, USA). TRIzol was purchased from Thermo Fischer Scientific (Wilmington, DE, USA). PrimePCR_PI3K-AKT signaling pathway (SABTarget list) H96 arrays, SYBR Green, and iScript advanced reverse transcriptase kit were purchased from Bio-Rad (Hercules, CA, USA). A propidium iodide DNA staining kit was purchased from Abcam USA (Cambridge, MA, USA). The Wes kit and reagents used were obtained from ProteinSimple (San Jose, CA, USA). The antibodies used were obtained from Cell Signaling Technology (Danvers, MA, USA) and ThermoFisher (Waltham, MA, USA).

Presence of angiogenic growth factors in EPC-CM was screened using a Human Angiogenesis Antibody Array (RayBiotech) as previously described [13] (link). The chemiluminescent signal of each factor on the array was acquired by ChemiDoc XRS (Bio-Rad) and the intensity measured by densitometry analysis using ImageJ (http://rsbweb.nih.gov/ij/).