Rporation (Paisley, UK). Production of cleaved ML-SA1 In Vivo C1-INH C1-Inhibitor was cleaved by incubation with trypsin Sepharose (30 mg/ml) for 6 h. The Sepharose was removed by centrifugation for three 2 min at 1000 g. Residual trypsin activityNIH-PA Author ManuscriptInnate Immun. Author manuscript; offered in PMC 2011 January 1.Thorgersen et al.Pagein the C1-INH containing supernatant was checked in a chromogenic assay using the chromogenic substrate S2222 (1 mM). Residual trypsin activity was 0.1 .NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSerum experiments Sera from 3 distinct pigs plus a serum pool of 20 humans had been pre-incubated for five min with C1-INH, iC1-INH, SPICE (pig experiments), compstatin (human experiments) or HSA. Escherichia coli was added to a final concentration of 108 bacteria/ml serum and incubated for 30 min at 37 . Complement activation was stopped by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of 20 mM. The Aztreonam medchemexpress samples have been kept on ice and then centrifuged for ten min at 1400 g (4), for removal of E. coli in the samples. The baseline sample (T0) was processed promptly. The sera were stored at -70 until analysed for complement activation. Whole-blood experiments Blood was collected from seven unique pigs (Sus scrofa domesticus, Landrace, out-bred stock) and six different humans. Each porcine and human blood was anticoagulated with lepirudin (50 g/ml) as described previously,17 and immediately split in 1.8-ml or 4.5 mlNunc tubes (Roskilde, Denmark) for incubation. The blood was made use of for complement, cytokine or flow cytometric analyses. For complement and cytokine analyses, blood was pre-incubated at 37 for five min with C1INH, iC1-INH, a complement inhibitor (SPICE in porcine experiments and compstatin in human experiments), or HSA. Escherichia coli was then added to a final concentration of 108 bacteria/ml entire blood inside the porcine experiments and 107 bacteria/ml whole blood in the human experiments. The samples were incubated for two h and four h. The baseline sample (T0) was processed straight away. Complement activation was stopped by adding EDTA to a final concentration of 20 mM promptly just after incubation. The blood samples had been centrifuged for 10 min at 1400 g (four) and also the plasma collected and stored at -70 until analysed. For the flow cytometry experiments, blood was pre-incubated at 37 for 5 min with C1INH, iC1-INH, a complement inhibitor (SPICE or compstatin), or HSA. Escherichia coli was then added to a final concentration of 108 bacteria/ml complete blood in each the porcine and human experiments plus the samples have been incubated for 10 min at 37 prior to analysis. Enzyme immunoassays The soluble terminal C5b-9 complement complex (TCC) was measured in an enzyme immunoassay (EIA), as described previously,21 and later modified.22 Briefly, the monoclonal antibody (mAb) aE11 reacting using a neoepitope exposed in C9 immediately after incorporation in the C5b-9 complicated was applied as capture antibody at a final concentration of 3 g/ml. A biotinylated anti-C6 mAb (Quidel Corporation, San Diego, CA, USA) was made use of as detection antibody at a final concentration of four g/ml. Each antibodies cross-react with pig and also the assay could be made use of to detect porcine TCC.22 The standard was regular human serum activated with zymosan and defined to include 1000 arbitrary units (AU)/ml. Zymosan-activated porcine serum was utilized as a constructive control. The buffer utilised as a diluent for the standards and samples and.
