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(ATCC #CCL‐119), >95% viability determined by eosin exclusion (Hoskins et al., ) on a representative sample: passaged twice weekly in T175 tissue culture flasks at a cell density of 0.5 to 2 × 106 viable cells/ml (see appendix 3B ; quality of assay

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analysis. Restriction enzyme buffer (10X) Dilute the 10X stock to 1.14X for Steps 12 and 13. RNase A, DNase-free (10 mg/mL; Sigma R6513) SDS (Sodium dodecyl sulfate; 20% w/v; Fisher BP166) Sodium acetate (3 M, pH 5.2

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a change in specific activity. 3.2. Preparation of Wnt3A CM for Fractionation 1. To the filtered CM from Section 3.1 , add Triton X-100 to1% (v/v), Tris-HCl, pH 7.5, to 20 mM, and NaN 3 to 0.01%(w/v) (e.g., to 930 mL CM, add 50 mL of 20% (v/v) TritonX