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		In vitro chondrogenesis of limb bud mesenchyme cells 1. Dissect limb buds from stage 22-24 chick embryos in sterile Howard's Ringers. > Do not use stage 25 or greater. 2. Wash with sterile Ca++, Mg++-free Tyrode's (CMFTy). 3. Pool limbs from 5 embryos in 10 ml 0.1% trypsin (Gibco 840-7072IL), 0.1% collagenase (Sigma C-6885) in CMFTy. Incubate at 37ºC withn gentle shaking for 1 hour. Gently pipet up and down with cut-off transfer pipet to disperse cells. Add 0.5 ml serum (FCS) to stop protease digestion. 4. Transfer to 10 ml tube. Centrifuge at 1500 rpm for 10 minutes. Discard supernatant. Gently flick to resuspend cells. 5. In sterile hood, add 10% FCS in CMFTy to 0.3-0.35 ml final volume. Remove small drop of cells. Leave remaining cells in hood. 6. Dilute 10 ul cells, 10 ul Trypan blue solution with 80 ul Howard's Ringer's. Count using hemocytometer. 7. Adjust cell concentration to 2 x 107cells/ml with 10% FCS in CMFTy. Transfer 15 ul drops to center of 8 wells of 24-well plate. Allow to set 1 hour. 8. Add 2 ml culture media (2% FCS in DMEM) to wells. Split into 5 groups of 3 wells each. Group 1 = control. Group 2 : add 20 ul 0.05 mg/ml poly-l-lysine. Group 3 : add 20 ul 0.1 mg/ml poly-l-lysine. Group 4 : add 20 ul 0.5 mg/ml poly-l-lysine. Group 5 : add 20 ul 0.5 mg/ml poly-l-lysine at 24 hours. 9. Culture for 3 days. Discard supernatant. Fix cultures by addition of 1 ml 4% paraformaldehyde. Store overnight at 4º C. 10. Wash with 2 x 1 ml PBS. Stain with 1 mg/ml Alcian blue in 0.1 N HCl overnight. Destain with 70% EtOH. Count the number of cartilage producing nodules for each well. Note: used Sigma collagenase, Worthington would probably work faster.