Europium conjugated streptavidin

MEF 10.1 Cells were grown in 24-well cell culture plates as described above. Each well was probed with 100 μL of biotinylated peptides at 1 μg/mL in cold DMEM/F12 with 0.1% BSA and incubated for one hour at 4°C. Following the incubation, cells were washed twice with ice cold PBS and fixed with 1.25% glutaraldehyde. Fixed samples were washed twice and stored in PBS for 24 hours. The samples were then blocked with 1% BSA in PBS and probed with 100 μL of Europium-conjugated streptavidin (Perkin-Elmer, Waltham, MA) in PBS/0.1% BSA for 30 min at RT. The plate was washed three times with PBS and enhancement solution added. The fluorescence counts of the control peptide (i.e., background), P5+14 treated cells, and P5+14 treated cells with added enzymes were measured using time resolved fluorescence on the Wallac Victor 3 (Perkin-Elmer) plate reader. Background counts were subtracted from all treatments. The percent reduction in bound peptide was calculated as 100%-(enzyme treated fluorescence counts/no enzyme treated counts x 100).

Pre-packed Exo-Spin size exclusion columns (Cell Guidance Systems) were washed with 6 mM EDTA in PBS, then 2 ml of PD effluent samples were added, and 15 × 1 ml aliquots were collected. RNA was extracted from 500 μl of each aliquot; the remainder was used for plate-based immuno-assays. Column fractions were bound overnight to protein-binding ELISA plates (Greiner Bio-One) and blocked with 1% (w/v) BSA in PBS. Bound material was detected with primary antibodies against CD9 (clone 209306; R&D Systems), CD15 (clone W6D3; BD Biosciences) or human serum albumin (clone 188835; R&D Systems), and developed using goat anti-mouse-biotinylated antibodies (Perkin Elmer) and europium-conjugated streptavidin (Perkin Elmer). Signals were measured by time-resolved fluorometry, using a Pherastar FS plate reader (BMG Labtech)^{32 (link)}.

Cytokine levels were analyzed by standard sandwich ELISA performed as per manufacturer’s instructions (BioLegend, San Diego, CA), modified to utilize europium-conjugated streptavidin (Perkin-Elmer), and detected with a Victor V3 plate reader (Perkin Elmer, San Jose, CA). Blocking experiments utilized antibodies to IFNγ (10μg/mL, BioLegend, San Diego, CA), IL-21 (10μg/mL, eBioscience, San Diego, CA), IL-22 (10μg/mL, eBioscience), CSF2 (10μg/mL, eBioscience), IL-9 (1μg/mL, eBioscience), IL-13 (2μg/mL, eBioscience), IL-4 (1μg/mL, eBioscience), IL-24 (2μg/mL, eBioscience), IL-3 (1μg/mL, eBioscience), Neuropilin-1 (1μg/mL, R&D Systems, Minneapolis, MN), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and corresponding isotype controls IgG1 and IgG2a (BioLegend, San Diego, CA). For CD124 blocking experiments, indicated cell types were incubated with 1μg/mL αCD124 at 4°C for 15 minutes, washed twice with PBS, then combined into co-culture. Supplementation assays were performed with recombinant mouse IL-2 and IL-4 (R&D Systems, Minneapolis, MN) at the indicated concentrations. For Luminex assays, media from indicated cultured populations were snap frozen in liquid nitrogen and sent to Eve Technologies (Calgary, Ontario, Canada) for mouse 32-plex and TGFβ 3-plex analysis.

Peptides with predicted binding to DR0401 (Supplementary Table 3) were synthesized (Sigma) and their binding to DR0401 was assessed through a competition assay, as previously described^{57 (link)}. Briefly, increasing concentrations of each citrullinated glucokinase peptide were incubated in competition with a biotinylated reference influenza hemagglutinin peptide (HA_306-318) at 0.02 μM in wells coated with DR0401 protein. After washing, residual biotin-HA_306-318 was detected using europium-conjugated streptavidin (Perkin Elmer) and quantified using a Victor2D time resolved fluorometer (Perkin Elmer). Curves were simulated using Prism software (Version 5.03, GraphPad Software Inc.) and IC₅₀ values calculated as the concentration needed to displace 50% of the reference peptide.