Bio plex pro human cytokines assay

Prehospital blood samples (20cc) were collected at 2 time points, i.) by prehospital providers while in the field and ii.) by ED staff upon arrival. The first sampling occurred when IV access was established by EMS, and samples were transported on ice at 0°C (Instant Cold Pack, Kimberly-Clark) for processing by the research team on arrival to the ED. Upon arrival in the ED, paramedics placed patients directly in a treatment room, where ED staff completed blood sampling during initial assessment. All blood sampling used pyrogen-free vacuum-evacuated heparin-, citrate-, and EDTA-containing Vacutainer® tubes. Research staff centrifuged samples at 4°C; separated plasma into aliquots; and stored samples frozen at −80°C until assay. We batch analyzed after a single freeze-thaw cycle. IL-6, IL-10 and TNF using the Bio-plex pro human cytokines assay (Bio-Rad Laboratories, Hercules, CA). All assays were measured on the Bio-Rad Bio-Plex® 200 System (Bio-Rad Laboratories, Hercules, CA). Blood processing and analysis were completed by Clinical Research, Investigation, and Systems Modeling of Acute Illness (CRISMA) Clinical Research Biospecimen Core. Research assistants were blinded to fluid group. Accepted upper limits of normal were: IL-6 5.9 pg/mL, IL-10 9.1 pg/mL and TNF 8.1 pg/mL, per the manufacturer’s specifications.^{18 (link)}

FLS were cultured in the presence or absence of spontaneously generated RA NETs for 3 days, washed twice with PBS to remove non-internalized NETs, then plated at a density of 100,000 cells/well in T-cell hybridoma medium or human T-cell medium. HC-gp-39 specific T cell hybridomas or human cit-vimentin specific T cells were added to FLS cultures at a ratio of 1:2 (FLS: T-cell) and incubated in presence or absence of 20 μg/mL of neutralizing antibodies against CD28 or MHCII for 5 days. Supernatants were then collected and centrifuged at 300 xg for 5 min at RT. Mouse IL-2 was quantified using ELISA Ready-SET-Go (eBioscience) for the hybridoma experiments and Bio-Plex pro-human cytokines assay (Bio-Rad) was performed for the human cit-vimentin T cell experiments, according to manufacturers’ recommendations. Aggrecan-specific T cell clones were used as negative control.