Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

Figure 1

RA fibroblast-like synovial cells proliferation in the presence of synovial fluid or TNFα. FLS were cultured in the presence of TNFα (25 ng/mL) or three SF pools (1 : 8 dilution in culture medium). The DNA synthesis was evaluated after 24 h incubation with 10 μM 5-ethynyl-2′-deoxyuridine (EdU). Cells were then fixed and permeabilized, and EdU incorporated into newly synthesized DNA was detected using the fluorescent Alexa Fluor 488 azide. Analysis was performed using a confocal microscope. Data were obtained from triplicate experiments. Results are shown as % of proliferation: % = (nuclei stained with Edu/total nuclei) × 100 and represent four independent experiments. Confocal microscopy images of a representative experiment are shown: cells cultured for 48 h in alone medium (a), TNFα (b), or SF pools (c, d, e), cells cultured for 72 h in medium alone (f), TNFα (g), or SF pools (h, i, j). Green staining: newly synthesized DNA, red staining: Hoechst staining of total DNA. versus FLS; versus FLS + TNFα.