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Haptotaxis is critical to cell guidance and development and has been studied in vitro using either gradients or stripe assays that present a binary choice between full and zero coverage of a protein cue. However, stripes offer only a choice between extremes, while for gradients, cell receptor saturation, migration history, and directional persistence confound the interpretation of cellular responses. Here, we introduce nanodot stripe assays (NSAs) formed by adjacent stripes of nanodot arrays with different surface coverage. Twenty-one pairwise combinations were designed using 0, 1, 3, 10, 30, 44 and 100% stripes and were patterned with 200 × 200, 400 × 400 or 800 × 800 nm 2  nanodots. We studied the migration choices of C2C12 myoblasts that express neogenin on NSAs (and three-step gradients) of netrin-1. The reference surface between the nanodots was backfilled with a mixture of polyethylene glycol and poly- d -lysine to minimize nonspecific cell response. Unexpectedly, cell response was independent of nanodot size. Relative to a 0% stripe, cells increasingly chose the high-density stripe with up to ~90% of cells on stripes with 10% coverage and higher. Cell preference for higher vs. lower netrin-1 coverage was observed only for coverage ratios >2.3, with cell preference plateauing at ~80% for ratios ≥4. The combinatorial NSA enables quantitative studies of cell haptotaxis over the full range of surface coverages and ratios and provides a means to elucidate haptotactic mechanisms.

Combinatorial nanodot stripe assay to systematically study cell haptotaxis