Elx405 washer

This assay was performed by the Human Immune Monitoring Center at Stanford University using a Mouse 48 plex ProcartaPlex™ kit (Thermo Fisher Scientific, EPX480–20834-901) according to the manufacturer’s instructions. Briefly, beads were added to a 96-well plate and washed in a Biotek ELx405 washer. Mouse EDTA Plasma samples were diluted 3-fold, added to the plate containing the mixed antibody-linked beads, and incubated overnight at 4 °C with shaking. Following overnight incubation, plates were washed in a Biotek ELx405 washer. Then biotinylated detection antibody was added for 1 h at ambient temperature (22°C) with shaking, followed by washing and the addition of streptavidin-PE, incubation for 30 min, and a final washing. Each sample was measured in duplicate on a Flex Map FM3D instrument with a lower bound of 50 beads per sample per cytokine and with the aid of custom Assay Chex control beads (Radix BioSolutions). Output MFI data were transformed to concentration in pg/ml using standards serially diluted and run in the assay. The resulting concentrations for cytokines/chemokines measured are reported here, and values below the detection limits in ≥33% of samples were excluded. Select cytokines’ differential protein abundance was compared using a two-sample Wilcoxon test in R v.3.6.3.

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.

Luminex was performed at the Stanford Human Immune Monitoring Core. Briefly, mouse 38 plex kits were purchased from eBiosciences/Affymetrix (Waltham, MA) and used according to the manufacturer’s recommendations with modifications as described below. Beads were added to a 96 well plate and washed in a Biotek (Winooski, VT) 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 was added for 75 minutes at room temperature with shaking. The plate was washed as above and streptavidin-PE was added. After incubation for 30 minutes at room temperature, wash was performed and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument or Flex3D with a lower bound of 50 beads per sample per cytokine. Custom assay control beads by Radix Biosolutions (Georgetown, TX) were added to all wells.

The assay was performed by the Human Immune Monitoring Center at Stanford University. Mouse 48-plex Procarta kits (EPX480-20834-901) were purchased from Thermo Fisher and used according to the manufacturer’s recommendations with modifications as described below. 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 overnight at 4 °C with shaking. Cold (4 °C) 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 biotinylated detection antibody was added for 60 min at room temperature with shaking. Plates were washed as described above 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 singlets. Plates were read on a FM3D FlexMap instrument with a lower bound of 50 beads per sample per cytokine/chemokine. Custom Assay Chex control beads were purchased from Radix BioSolutions, and were added to all wells.