Thrombin induces synovial proliferation and progression to S phase in RA SFs. Cells were cultured for 48 hours in DMEM containing 1% FCS and then stimulated with the indicated amount of thrombin. For the proliferation assay, at 24 hours after thrombin stimulation, cells were stained with TetraColor One including tetrazolium and electron-carrier mixture for detecting cell proliferation. The optical density (OD) was measured by ELISA plate reader at 450 nm. For analysis of the cell cycle, at 24 hours after thrombin stimulation, cells were collected, washed with PBS, and fixed in 70% ethanol for 2 hours at 4°C. After treatment of cells with 10 μg/ml ribonuclease for 15 min at 37°C, fixed cells were stained with 50 μg/ml propidium iodide for 2 min. The DNA content was subsequently measured by FACScan fluorescence-activated cell sorter. (a) Dose-dependent proliferation of synovial fibroblasts (SFs) from rheumatoid arthritis (RA) patients. The OD was measured by ELISA plate reader at 450 nm. (b) Histogram representing the cell cycle in RA SFs, as detected by FACScan. (c) Dose-dependent S-phase progression of the cell cycle in RA SFs. Numbers represent the percentage of cells exhibiting mean channel fluorescence (FL2-H) in the S/G2/M phase of the cell. Data are expressed as mean ± standard deviation for five experiments, using five independent donors. **P < 0.01 in comparison with the value found without thrombin stimulation.
Nakayamada et al. Arthritis Research & Therapy 2005 7:R476-R484 doi:10.1186/ar1694