Human 63 plex kits

Levels of circulating cytokines in the blood were measured using a 63-plex Luminex antibody-conjugated bead capture assay (Affymetrix) that has been extensively characterized and benchmarked by the Stanford Human Immune Monitoring Center (HIMC). Human 63-plex kits were purchased from eBiosciences/Affymetrix and used according to the manufacturer’s recommendations with modifications as described below. Briefly, beads were added to a 96-well plate and washed using a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 h followed by overnight incubation at 4°C with shaking. Cold and room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation, plates were washed using a Biotek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. The plate was washed as describe earlier and streptavidin-PE was added. After incubation for 30 min at room temperature, a wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay control beads by Radix Biosolutions were added to all wells.

Levels of cytokines in the supernatants from human primary CD14⁺ monocytes and Kupffer cells were measured using a 63-plex Luminex antibody-conjugated bead capture assay (Affymetrix). This assay was performed in the Human Immune Monitoring Center at Stanford University. Human 63-plex kits were purchased from eBiosciences/Affymetrix and used according to the manufacturer’s recommendations with modifications as described below. Briefly, beads were added to a 96 well plate and washed in a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 hour followed by overnight incubation at 4°C with shaking. Cold and Room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation plates were washed in a Biotek ELx405 washer and then biotinylated detection antibody added for 75 minutes at room temperature with shaking. Plate was washed as above and streptavidin-PE was added. After incubation for 30 minutes at room temperature wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay Control beads by Radix Biosolutions are added to all wells.