Prosense 750ex

Mice were injected IV with 4 mg of ArthritoMab monoclonal antibody cocktail (MD Biosciences), and then IP 3 days later with 100 μg LPS (MD Biosciences). In some experiments, 0.7 mg of anti-MICL or isotype control antibodies were administered IP every 24 h from day 4 to day 13. Daily clinical scoring was undertaken using a scale of 0 (no visible signs of redness or swelling) to 4 (extensive swelling with signs of deformity). To generate radiation chimeras, animals received 2×500 rad (IBL437C irradiator, Cis Bio International) and were then reconstituted with 2–4×10⁶ bone marrow cells from donor mice. For histology, joints were isolated, fixed, decalcified, sectioned and stained with H&E. In vivo imaging was performed using a Bruker in vivo MS FX Pro imager, after a single IV administration of OsteoSense 680EX (NEV10020EX, PerkinElmer, UK), 24 h before the start of the collagen antibody-induced arthritis (CAIA) protocol. Twenty-four hours before image acquisition, animals were also injected IV with ProSense 750EX (NEV10001EX, PerkinElmer, UK).

Inflammation-associated factors were monitored by FMT in vivo after 2 months. FMT enables real-time three-dimensional quantitation of fluorochrome distribution in tissues of live animals [27 (link)-30 (link)]. The mice were injected with a single dose of ProSense 750 EX and MMPSense 680 fluorescent imaging agents (PerkinElmer, Waltham, MA, USA) 24 hours before imaging. ProSense detects cathepsins B, L, and S and plasmin. MMPSense detects MMP-2, MMP-3, MMP-9 and MMP-13 activity. Mice were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (1 mg/kg), placed in an upright position in the imaging chamber and then imaged using the VisEn FMT Optical Imaging System (PerkinElmer). A near-infrared laser diode emitting continuous wave radiation at wavelengths of 670 nm or 746 nm transilluminated the lower body of each animal from posterior to anterior, and both excitation and emission signals were detected by a charge-coupled device camera and appropriate band-pass filters. The no TM-PMM group (n = 12), TM-PMM group (n = 8) and TM-sham group (n = 3) were imaged. The picomolar concentrations of probes in the knee joint were determined using region of interest analysis. We used FMT imaging to confirm that Ihh deletion decreases inflammatory mediators in vivo, then compared the results with histological scores and molecular studies.