Podoplanin is expressed in cultured primary FLS and the expression is increased by pro-inflammatory cytokines. (A) Immunofluorescence staining of primary rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) showing podoplanin (red) and actin stress fibres (green). Note accumulated podoplanin staining in membrane protrusions (arrowheads). (B) Magnification of podoplanin-positive RA-FLS. (C and D) Flow cytometry (FACS) of primary FLS cultures from patients with RA (filled bars) and patients with osteoarthritis (OA) (striped bars) showing the percentage of positive cells of viable cell populations using (C) antipodoplanin and phenotype markers (CD90, CD68 and CD29) and (D) cadherin-11 antibodies. (E) Immunocytochemistry of an aggressively growing RA-FLS culture using antipodoplanin antibody. Note the dendritic phenotype with long cytoplasmatic protrusions. (F) Representative flow cytometry plot of primary RA-FLS stained for podoplanin and α-smooth muscle actin (α-sma) showing the double-positive population (podoplanin+ and α-sma+) of 58.2% in the upper right quadrant. (G) Graph showing the percentage of podoplanin-positive primary FLS by flow cytometry at baseline, 24 and 48 hours of stimulation with control (complete medium) (open circles), 10 ng/ml tumour necrosis factor (TNF)-α (filled squares), 1 ng/ml interleukin (IL)-1β (open diamond) and 1 ng/ml transforming growth factor β receptor 1 (TGF-β1) (filled triangles), respectively, of a representative culture. The experiment was run in triplicate and repeated four times using different OA-FLS and RA-FLS cultures, which showed similar results but starting at different baseline levels of podoplanin expression.