Recombinant il 10

Neutralising experiments were performed to investigate the role of increased circulating IL-10 in mediating post-operative changes in mHLA-DR cell surface expression. Either anti-IL-10 (10ng/mL, R&D systems, UK) or control non-specific goat Immunoglobulin G (IgG) (10ng/mL, R&D systems, UK) was added to the culture media and incubated with peri-operative serum as previously described [3 (link)].
In order to investigate the role of IL-10 alone, recombinant IL-10 experiments were also performed. Pooled healthy PBMCs were cultured in duplicate with 30% pre-operative patient serum and incubated with recombinant IL-10 (10ng/mL, R&D systems, UK), mHLA-DR antigen density was then quantified as described previously by flow cytometry.
Previous trials in sepsis [26 (link)] and trauma [27 (link)] have demonstrated ex vivo mHLA-DR cell surface re-expression following systemic treatment with immune-stimulants such as GM-CSF or IFN-γ. Stimulation experiments were therefore performed in which IFN-γ (R&D systems, UK) or GM-CSF (R&D systems, UK) were added to the culture media and incubated with peri-operative serum [3 (link)]. mHLA-DR antigen density was then quantified as described previously by flow cytometry.

The concentrations of cytokines were measured by sandwich enzyme-linked immunosorbent assay. Abs directed against human IL-10 and biotinylated anti-human IL-10 (R&D Systems) were used as the capture and detection antibodies, respectively. The concentration of cytokines present in the test samples was determined from standard curves established with serial dilutions of recombinant IL-10 (R&D Systems). The absorbance at 450 nm was measured using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).