Role of S100A9 in Arthritis activities of S100A9 and investigate the impact of anti-S100A9 therapy on acute arthritis development. by western blot (Fig. 1 B). Clone 2A5 used in all in vivo experiments recognized only murine S100A9. Materials and Methods Protein and Antibody Production Murine S100A9 monoclonal antibody (mAb) was generated using the ClonaCellH – H hybridoma cloning kit (Stemcell Technologies) according to the manufacturer’s instructions. Briefly, Sprague Dawley rats (Charles River) were immunized four times with full length murine S100A9 protein. Spleen and myeloma cells were mixed and plated on a methylcellulose-based selection medium. Individual colonies were picked using a pipette, transferred into a 96-well plate and tested for mAb secretion. Clone 2A5 (IgG1k) was selected based on its ability to inhibit recruitment in vivo in response to murine S100A9. Clone 2A5 was cultured in CeLLineTM flasks (BD Biosciences) for Ab production. The supernatant was collected after 14 days and purified using the PierceH Thiophilic Adsorbent method. Purified rabbit IgGs directed against murine S100A9 and S100A8 and recombinant human S100A9 (rhS100A9) were prepared as previously described [14]. The absence of endotoxin contamination in Ab and protein preparations was confirmed using the Limulus amebocyte assay (Cambrex) for Abs and proteins before use. Antibody concentra- tion for clone 2A5 was determined by Bradford protein assay (Bio- rad). Specificity of the murine monoclonal antibody was confirmed Induction of LPS-CIA All experiments were carried out in accordance with the Laval University animal protection committee (Sainte-Foy, Quebec, Canada). Six- to 8-week-old female DBA/1 mice, obtained from Jackson Laboratories were immunized subcutaneously at the base of the tail with chicken collagen type II (100 mg/mouse, Chondrex inc) emulsified in complete Freund’s adjuvant, prior to receiving an i.p. injection of LPS (25 mg/mouse) 27 days later according to the manufacturer’s instructions (Fig. 1A). Disease activity was monitored every other day on a scale of 0 to 4 per paw (0, normal; 1, mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2, moderate redness and swelling of ankle of wrist; 3, severe redness and swelling of the entire paw including digits; 4, maximally inflamed limb with involvement of multiple joints) for a maximum disease score of 16 per mouse. To evaluate the effects of S100A9-neutralizing Abs on LPS-CIA development, mice were injected with 2 mg of Abs 24 h before LPS injection, and then injected twice a week with 1 mg of Abs until the end of the protocol. An anti- TNFa (clone MP6-XT3, Upstate Cell Signaling) and a rat isotype control treatment (ChromPure Rat IgG, whole molecule, Jackson Figure 1. LPS-CIA model. (A) Mice (25 mice/group) were immunized at the base of the tail with 100 mg of chicken type II collagen on day ‘‘227’’ and then injected i.p. with LPS (25 mg in PBS) on day 0. Two milligrams of neutralizing Abs or isotype control Abs were injected into mice 24 h before LPS injection. During the protocol, five injections were given, at 2, 5, 8, 12, and 16 days post-LPS injection. On day 5 and 19, mice were sacrificed to collect the paws and the serum for further analysis. (B) Murine monoclonal anti-S100A9 (Clone 2A5) was tested for specificity by western blot against murine protein mS100A9, mS100A8, a lysate of murine neutrophils, human protein hS100A12, hS100A9, hS100A8 and a lysate of human neutrophils. doi:10.1371/journal.pone.0045478.g001 PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45478