In vivo cell migration assay

The effects of nitration of CXCL12 on the ability of CXCL12 forms to attract lymphocytes in vivo by extravasation from the blood circulation into the tibiofemoral articulation were examined by intra articular (i.a.) injection of the different CXCL12 forms in 8 weeks old wildtype C57BL/6 mice. Three days prior to the experiment, the water of the mice was changed to a 1.7mg/ml solution of the CD26 inhibitor sitagliptin [Merck Sharpe & Dohme (MSD), Whitehouse Station, NJ)] in water to partially block the activity of this enzyme for which CXCL12 is an efficiently cleaved and inactivated substrate [57]. Endotoxin levels in the injected samples were tested with the Limulus amoebocyte lysate test (Cambrex, East Rutherford, NJ) and were lower than 0.125 pg LPS per μg chemokine. Different concentrations of the chemokine forms, diluted in a 0.9% (w/v) NaCl solution were injected i.a. as described previously [100], mice were placed under anaesthesia using 3.75% (w/v) ketamine, 0.25% (w/v) xylazine in PBS and injected i.a. with 10μl of the chemokine dilutions. After 3 hours of incubation, the mice were sacrificed and the articular cavity was washed using 3% (w/v) BSA in PBS. Total leukocytes were counted using a Neubauer chamber and Turk's staining solution. Hereafter, the samples were counted differentially on May-Grünwald-Giemsa stained Cytospins (Shandon III). Experiments were performed in the animalia of the University of Minas Gerais and Leuven. All experiments using laboratory animals were reviewed and approved by the Animal Ethical Committee of the University of Minas Gerais and the Animal Ethical Committee of the University of Leuven.