Bio plex protm human chemokine assay

Cell free ascites samples were analyzed for CCL2/MCP-1, CCL17, CCL22, CXCL1/Gro-α, CXCL8/IL-8, CXCL9, CXCL10, CXCL11 and CXCL12/SDF-1 using a multiplex Bio-Plex Pro^TM Human Chemokine assay (BioRad, #171AK99MR2), and CCL5/RANTES Bio-Plex Pro Human Cytokine Assay (BioRad #171B5025M). Samples were prepared according to manufacturer's recommendations. In short, 50 µl of freshly thawed samples and prepared standards were transferred to designated wells in the 96-well plate, with each well containing 50 µl magnetic bead solution. The plate was incubated on a shaker for 1hr (850 rpm, the setting for all following incubation). Next, 25 µl detection antibody was added to each well and the plate was incubated on shaker for 30 min. in dark. Last, 50 µl streptavidin was added and the plate incubated for 10 min. on shaker in dark. The plate was washed 3 times between all incubation steps.
The samples were acquired on a Bio-Plex 200 system (BioRad, CA, USA) and analyzed with Bio-Plex Manager^TM v.6 software.

To determine the concentration of cytokines and chemokines secreted by the microtissues, we harvested and pooled 24 single co-cultivated microtissues which were stored at −80°C until use. Concentrations were determined using ProcartaPlex™ Essential Human Th1/Th2 immunoassay (Affymetrix, eBioscience) and Bio-Plex ProTM Human Chemokine Assay (Bio-Rad, USA), following manufacturers’ instructions. Samples were measured using a Magpix (Luminex) and data were analyzed by the “xPONENT 4.2” software (Luminex).