Streptavidin pe

EDTA-plasma was prepared by immediately placing the blood sample on crushed ice. Within 30 minutes samples were centrifuged at 2500 × g for 15 minutes at 4 °C and plasma immediately stored at −70 °C. Complement activation was measured as the soluble TCC (sC5b-9) using multiplex xMAP technology (Bio-Plex; Bio-Rad Laboratories, Inc., Hercules, CA, USA) as previously described [21 (link)]. Briefly, mouse anti C5b-9 Ab (clone aE11; Diatec Monoclonals AS, Oslo, Norway) was coupled to carboxylated magnetic beads (Bio-Plex Pro) using a Bio-Plex amine coupling kit (Bio-Rad Laboratories, Inc.). Coupled beads were then incubated with samples, followed by biotinylated anti-C6 monoclonal antibody (mAb) (Quidel, San Diego, CA, USA) and streptavidin-PE (Bio-Rad Laboratories, Inc.). The international complement standard number 2, recently described [22 (link)], was used as the calibration curve. Measurement and data analysis was performed with the Bio-Plex™ 200 system and the Bio-Plex Manager software version 6.0. Porcine cytokines (n = 9) were analyzed in plasma using a multiplex kit from Bio-Rad Laboratories, Inc. according to the manufacturer’s instructions. The kit comprised the following cytokines: interferon (IFN)-α, IFN-β, tumor necrosis factor (TNF), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and IL-12p40.

Sagittal sections of whole knee joints in the area of the defect were labeled using a standard TRIS buffer immunohistochemistry protocol to identify the MRL-specific marker Class 1 MHC H2Kk. Briefly, sections were deparaffinized, then washed and permeabilized in TRIS buffer / 0.1% Triton-X 100 for 45min (all Sigma). Antigen retrieval (Proteinase K, 30min at 37° C, Life Technologies), blocking (10% normal goat serum and 1% bovine serum albumin, both from Sigma) and endogenous biotin blocking steps (15min egg white, 10% w/v in water and 15 min skim milk blocking, all from Sigma) were performed and the sections were incubated overnight at 4°C with a biotin-conjugated primary antibody to Class 1 MHC H2Kk (1:200; Abcam). The primary antibody was visualized using streptavidin-HRP (1:500 for 2h at room temperature, Sigma) followed by exposure to DAB (Sigma), 2.5-3min, or by streptavidin-PE (1:500 for 2h at room temperature, Bio-Rad).

The cells obtained from whole blood were washed twice in PBS with 2% FCS before staining with either anti‐CD11b‐FITC (Abcam) or anti‐CD41‐FITC (eBioscience) with biotinylated anti‐HLL followed by Streptavidin‐PE (Bio‐Rad). Cells were then washed twice before analysis on a BD FACSCALIBUR with CellQuest Pro software. Erythrocytes were identified by forward/side scatter profile.
For viability assays, cells were washed twice with PBS and then incubated with Fixable LIVE/DEAD Near‐IR fluorescent reactive dye (Thermo Fisher Scientific, Paisley, Renfrewshire, UK) for 30 minutes at 4°C. Cells were washed, fixed for 15 minutes in 1% paraformaldehyde, then washed with PBS‐5% FCS and stored at 4°C before acquisition and analysis within 24 hours on an LSRII/Fortessa flow cytometer at the BRC Flow Cytometry Laboratory, King's College London with FlowJo software (Treestar Inc). Macrophages identified by forward/side scatter profile.