Flow cytometry
The antibodies presented in Table 3 were used for FACS analyses of primary-culture RA and OA synovial cells (following 7 days of culture), isolated RA-SFB and OA-SFB (immediately following isolation or at fourth-passage), conventional fourth-passage RA-SFB and OA-SFB, normal skin-FB (primary-culture or fourth-passage), or repeated-passage endothelial cells (HUVEC). Primary antibodies were used at concentrations of 10–20 μg/ml. Standard single and double-staining procedures for surface molecules were performed as previously described [10]. The specificity of staining was confirmed using isotype-matched control mAbs, rabbit serum, or rabbit Ig at identical concentrations (Table 3). The FACS analysis in RA-SFB and OA-SFB was not always performed with the complete set of antibodies due to the initial establishment of the isolation procedure.
Trypsinized cells were washed twice with serum-free medium and fixed for 10 min at 4°C in 4% paraformalde-hyde (Fluka, Deisenhofen, Germany) for detection of antigens in cytoplasm and/or nucleus (eg prolyl-4-hydroxylase; Table 3). After washing twice in PBS/2% FCS, the pellet was resuspended in permeabilization buffer (PBS/1% FCS, 0.01% NaN3, and 0.5% saponine; Serva, Heidelberg, Germany) and incubated for 10 min at room temperature. Unlabeled primary mAbs were added at saturating concentrations and detected with a secondary phycoerythrine-labeled goat anti-mouse antibody (Dako), both for 45 min at 4°C in permeabilization buffer. Polyclonal antibodies were incubated with a secondary mouse anti-rabbit antibody (Dako) before using the phycoerythrine-labeled goat anti-mouse antibody. After washing twice in permeabilization buffer, the cells were incubated for 10 min in PBS/5% FCS and 0.1% NaN3 without saponine. In the special case of the CD68 epitope recognized by the mAb PG-M1, present on the cell surface and in the cytoplasm, FACS analysis was performed in both non-permeabilized and permeabilized cells. For double-staining analyses, fluoresceine isothiocyanate (FITC)-labeled Anti-Thy-1 mAb AS02 (20 μg/ml in PBS/2% FCS) or a matched concentration of the FITC-labeled IgG1 isotype control mAb were added for 30 min at 4°C.
Analyses were performed on a FACSCAN® using the software Cell Quest (Becton Dickinson, San Jose, CA, USA). Forward and side scatter gates were set to include all viable cells. In single-staining experiments, a gate was set to exclude 99% of the cells stained with control Ig. The gates for control Ig in double-staining experiments were placed to limit, to less than 1%, not only the individual percentages in the upper left, upper right, and lower right quadrants, but also the sum of the percentages in the upper left and upper right quadrant, or in the upper right and lower right quadrant.