Human FLS (as control), RA patient FLS and synoviocyte growth medium were purchased from Cell Applications (Santiago, California, USA) [31 (link)]. FLS were cultured at 37 °C in 5% CO2 in synoviocyte growth medium containing 100 μg/mL streptomycin and 100 U/mL penicillin. FLS in the exponential phase of growth were seeded into 10-cm dishes (1 × 106 cells/dish).
After FLS had spread across the dishes, they were fixed for 15 min in 2% paraformaldehyde, blocked for 1 h with rabbit serum (Sigma), then incubated for 1 h with antibody against the FLS marker vimentin (1:50; Abcam, Cambridge, MA, USA). The dishes were washed three times with 0.01% saponin in phosphate-buffered saline (PBS) for 15 min each, then incubated for 1 h with secondary antibody conjugated with fluorescein (Jackson Immuno Research, West Grove, PA, USA). Dishes were washed in PBS, the nuclear stain DAPI was added, the coverslips were washed three times with 0.01% saponin in PBS for 15 min each, and then they were washed twice in PBS for 10 min each. The dishes were mounted on slides using anti-fade mounting medium and analyzed under an IX2-ILL100 fluorescence microscope (Olympus, Tokyo, Japan).
After FLS had spread across the dishes, they were fixed for 15 min in 2% paraformaldehyde, blocked for 1 h with rabbit serum (Sigma), then incubated for 1 h with antibody against the FLS marker vimentin (1:50; Abcam, Cambridge, MA, USA). The dishes were washed three times with 0.01% saponin in phosphate-buffered saline (PBS) for 15 min each, then incubated for 1 h with secondary antibody conjugated with fluorescein (Jackson Immuno Research, West Grove, PA, USA). Dishes were washed in PBS, the nuclear stain DAPI was added, the coverslips were washed three times with 0.01% saponin in PBS for 15 min each, and then they were washed twice in PBS for 10 min each. The dishes were mounted on slides using anti-fade mounting medium and analyzed under an IX2-ILL100 fluorescence microscope (Olympus, Tokyo, Japan).