1 2 3 4 5 6 7 8

		Experimental Design 1. Dissect cranial facial regions, wing buds and hind limb buds from HH stage 22-24 chick embryos in sterile Howard's Ringers solution. Do not use stage 25 or greater. 2. Wash with sterile Ca++, Mg++-free Tyrode's (CMFTy) 3. Pool facial cells and limbs from 5 embryos in 10ml 0.1% trypsin (Gibco 840- 7072IL), 0.1% collagenase (Sigma C-6885) in CMFTy. Incubate at 37oC with gentle shaking for 1 hour. Gently pipette up and down with cut off transfer pipette to disperse cells. Add 0.5 ml newborn calf serum (NCS) to stop protease digestion. 4. Transfer to 10ml tube Centrifuge at 1500 rpm for 10 minutes. Discard supernatant. Gently flick to resuspend cells. 5. In sterile hood, add 10% NCS i n CMFTy t o 0. 3 - 0.35 ml final volume. Remove small drops of cells. Leave remaining cells in hood. 6. Dilute 10ml cells, 10ml Trypan blue solution with 80ml Howard's Ringer's. Count using hemocytometer. 7. Adjust cell concentration to 2 x 10^7 cells/ml with 10% NCS in CMFTy. Transfer 15 micro liter drops to center of wells on a 24 well plate. Allow to set 1 hour.