Milliplex immunoassay

At 16, 32, and 64 hours post-agonist exposure, 200 µl was removed from GE KER, DC, and HTL single cell cultures and GE KER, DC, and HTL multi-cell cultures. 200 µl of LGM-3 was added back. At 64 hours, cell culture media was removed from each transwell layer, filtered, and frozen at −80 °C until analysis. CCL3 (MIP1α), CCL4 (MIP1β), CCL5 (RANTES), CSF2 (GM-CSF), IL12(p40), IL1α, IL6, IL8, TNFα, and VEGF concentrations were determined using Milliplex immunoassays (Millipore, Billerica, MA, USA) routinely run in our laboratory^{28 (link),29 (link)}.
At 64 hours post incubation, all cell culture media was removed from the MM and DC multi-cell culture, filtered, and frozen at −80 °C until analysis. MM cell lines and DC were collected and lysed. Concentrations of IL6, IL10, VEGF, and TGFβ1 were determined in the cell culture media using Milliplex immunoassays (Millipore, Billerica, MA, USA) and concentrations of PD-L1, CD47, IDO, and FASL in cell lysates were determined using enzyme-linked immunosorbent assay kits (ELISA, American Research Products, Inc., Waltham, MA).
Standard curves for each cytokine were prepared from 0.64 to 10,000.00 pg/ml and concentrations of chemokines and cytokines in each sample were interpolated from standard curves (MILLIPLEX Analyst v5.1, Millipore, Billerica, MA or xPonent v3.1, Luminex, Austin, TX).