Bioplex 200 luminex bead array reader

A total of 34 cytokine and angiogenic factors (Supplementary Table S1) were assessed in pre- and post-intervention serum using multi-spot ELISA-based assays on the Meso Scale Discovery (MSD) Sector Imager 2400 (Meso Scale Discovery, Rockville, MD, USA) using either a 30-plex human cytokine bead-based assay (Invitrogen, Carlsbad, CA, USA) on a Bio-Plex 200 Luminex bead array reader (Bio-Rad, Hercules, CA, USA) or Quantkine colorimetric sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA). Briefly, assay beads were washed and added to pre-wetted 96-well plates. Incubation buffer was added to each well and 100 μl of standard curve proteins or 100 μl of diluted patient sample serum was added to appropriate wells. Plates were incubated for 2 h at room temperature on an orbital shaker and then washed. A 100 μl volume of biotinylated detector antibody was then added to each well, the plate reincubated for 1 h at room temperature on an orbital shaker, and then washed. A 100 μl volume of streptavidin-RPE was added to each well, then the plate was incubated for 30 min at room temperature on an orbital shaker, and then washed again. The plate was read on a Luminex Bio-Plex 100 liquid suspension array bead detector (Bio-Rad). All samples were run in duplicate and analyte concentrations derived using a five-parameter logistic model fit to the standard concentration curves.