Human fibronectin coated plates

Peripheral blood sample (20 ml) was collected and centrifuged using the Ficoll-density gradient method. Buffy coat mononuclear cells (MNCs) were collected and washed with HANKs’ balanced salt solution twice. MNCs were then resuspended with EGM-2 medium (Lonza, San Diego, CA) and plated on human fibronectin-coated plates (BD Biosciences, Bedford, MA) at a density of 4 × 10⁶ cells. Culture medium was changed every 3 days, and passage was performed when cell colonies were formed [12 (link), 13 (link)].

Reverse transfection of indicated siRNAs was performed with RNAiMax (Invitrogen) according to manufacturer’s protocol in indicated media without antibiotics. Briefly, 60pmols of siRNA was incubated with 1μl RNAiMax for 20 minutes at room temperature. Rat limb bud cells were plated in 12-well human fibronectin-coated plates (BD Biosciences) at a density of 1.5x10⁵ cells/well after siRNA complex formation such that final total siRNA concentration was 50nM. The following siRNA constructs were used (Ambion/Thermo Fisher Scientific): Control #1 (catalog 4390843), Stat1 (ID s129044), Stat3-1 (ID s129046), Stat3-2 (ID s129047), Stat5A (s128672) and Stat5B (ID s129049).