Flow Cytometric Analysis of Synovial Cells

Immediately after euthanasia, the skin of the hind limbs was removed and synovial tissues around the knee joints were pooled from 4 animals per group as previously described^{34 (link)–36 (link)}. The harvested tissues were minced using sterile scalpel and digested in a 1 mg/ml collagenase type I (Sigma) for one hour at 37 °C and rinsed through a 70-µm filter (BD Biosciences, North Ryde, NSW, Australia)^{34 (link)–36 (link)}. The cells were suspended in phosphate-buffered saline (PBS) containing 20 µg/ml of antibodies. Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- or allophycocyanin (APC)-conjugated antibodies against mouse F4/80, CD11b and MHCII was obtained from BD Biosciences. For isotype controls, FITC-, PE-, APC- conjugated nonspecific rat immunoglobulin G was substituted for the primary antibody. After incubating with antibody cocktails for 30 minutes at 4 °C, the cells were washed with PBS and resuspended in PBS for the analysis. Data were acquired on a BD Accuri C6 cytometer (BD Biosciences, North Ryde, NSW, Australia) and analysis of the data was performed using BD Accuri C6 1.0 software (BD Biosciences, North Ryde, NSW, Australia).