Persistence assay for assessing the formation of a chemoattractant concentration gradient. (A) Schematic illustration of the method of the persistence assay. Ten μl of chicken liver extract were placed in the center of one quadrant (Zone 4) of the assay chamber containing 2 ml of 0.1% low melting point agar solution. Zero, 5, or 10 min later, 3 μl of the agar solution was transferred from point 1 or point 2 to the center of a quadrant (Zone 1) of a fresh chamber, and then a planarian was placed in Zone 1. Planarians normally move around continuously after transfer to an assay chamber or culture dishes, and therefore the time spent in Zone 1 would normally gradually decrease. However, they remain in the initial region (Zone 1) if a sufficiently high concentration of chemoattractant is present there. (B) Time spent in Zone 1 during 1-min assay period. The chemoattractant diffused from Zone 4 to Zone 1 within 10 min after it was added.