Magpix multiplexing system

Serum cytokine and chemokine levels in serum were measured using a Cytokine Milliplex Assay Kit and a MAGPIX Multiplexing System (both from MilliporeSigma) following the manufacturer’s protocol.

MDA-MB-231 breast cancer cells were cultured in 10 cm dishes in 1.8 × 10⁶ cells per mL and incubated for 24 h at 37 °C. Before compound treatment medium was changed to serum-free medium. Cells were treated with PsA-D for 15 min, followed by incubation with 1 µg/mL LPS. Afterwards, cells were lysed with the lysis buffer provided in the NF-κB magnetic bead kit from Merck Millipore (Darmstadt, Germany) to obtain phosphorylated proteins from the nucleus. Protein concentration was determined with Bradford reagent (Roth, Karlsruhe; Germany). Samples were diluted to achieve a concentration of 0.8 mg/mL of total proteins. The subsequent protocol was according to manufacturer’s instructions.
MDA-MB-231 breast cancer cells and were seeded in 96-well plates in 4 × 10⁵ cells per mL and MDA-MB-453 in 6 × 10⁵ cells per mL and incubated for 24 h at 37 °C. THP-1 cells were seeded in 4 × 10⁵ cells per mL and after 1 h of incubation differentiated with 10 ng/mL PMA for 24 h. Cells were treated with PsA-D for 20 min and afterwards with 1 µg/mL LPS for 24 h. Supernatant was harvested and stored at −20 °C until measurement of cytokines. The subsequent protocol was performed according to the manufacturer’s instructions with the MAGPIX^® Multiplexing System from Merck Millipore (Darmstadt, Germany).