Flour s max multi imager

RayBiotech human cytokine antibody arrays were used to study the effect of butein on 60 cytokine proteins released by TNF-α-activated TNBC cells. Each experiment was performed in triplicate and according to the manufacturer’s instructions. Shortly, antibody-coated array membranes were first incubated for 30 min with 1 ml of blocking buffer. Then, blocking buffer was decanted and replaced with 1 ml supernatant from cells exposed to the different treatments for a 24-h period. Treatments consisted of control (cells + DMSO) samples, cells treated with butein (5 μM), TNF-α (40 ng/ml), and the combination of butein (5 μM) + TNF-α (40 ng/ml). Membranes were incubated overnight at 4°C with mild shaking. The next day, the media were decanted; membranes were washed, and subsequently incubated with 1 ml biotin-conjugated antibodies for 2 h. Lastly, biotin-conjugated antibodies were removed, and membranes were washed again and incubated with HRP-conjugated streptavidin for 2 h. In this assay chemiluminescent reagent was used and the image of spots was captured using a Flour-S Max Multi-imager (Bio-Rad Laboratories, Hercules, CA, USA), and the spot density was determined with Quantity One Software (Bio-Rad Laboratories, Hercules, CA). Excel-based data analysis was performed, using Human Cytokine Array software C1000 (CODE: S02-AAH-CYT-1000) from RayBiotech.

PGG effect on pro-inflammatory cytokines released by TNBC MM-231 and MM-468 cells activated by TNF-α after 24-h treatment was studied using human cytokine antibody arrays. Experiments were carried out in triplicate. Following the protocol, 1 ml of blocking buffer was incubated with the arrays coated with the antibody and then replaced with 1 ml of supernatants of different samples as follows: control (cells + DMSO) samples, PGG-treated cells (6.25 and 25 μM for MM-231 and MM-468 TNBC cells, respectively), TNF-α (50 ng/ml), and the combination of PGG (6.25 or 25 μM) + TNF-α (50 ng/ml). Following overnight incubation at 4 °C, arrays were washed, and 1 ml of biotin-conjugated antibodies was added. After 2 h-incubation, HRP-conjugated streptavidin was added and left for 2 h. Lastly, with the addition of a chemiluminescent reagent, the image of spots were taken (Flour-S Max multi-imager- Bio-Rad Laboratories, Hercules, CA, USA), and the density of spots determined (Quantity One Software- Bio-Rad Laboratories, Hercules, CA).