Femtotip microcapillary

A total of 1 × 10⁵ developed cells were seeded on a 27-mm glass-bottom dish (Iwaki). cAMP gradients were produced by a Femtotip microcapillary (Eppendorf) containing 100 μM cAMP and ATTO 633 (AD 633–21, ATTO-TEC) under a constant pressure of 10 hPa using a FemtoJet (Eppendorf). Images were acquired at 10-s intervals by confocal microscopy (A1, Nikon). Cells moving separately (i.e., cells not in contact with other cells) were tracked using G-Count (g-angstrom.com). Each trajectory was divided into short trajectories of 1-min intervals. The chemotaxis index and motility speed were analysed for each short trajectory. The chemotaxis index was the cosine of the angle formed by the intersection of the line connecting the starting and end points of movement with the line connecting the starting point and the micropipette. Motility speed was the total travelled distance divided by time. The analysed values of the trajectories were sorted by the distance from the end point of each trajectory to the tip of the micropipette and are shown as bar graphs.

Five microliter of cell suspension, at a density of 4 × 10⁷ cells mL⁻¹, was deposited on 2% water agar with or without 1 mM EGTA and incubated at 21 °C for 12–15 h. Following slug formation, a piece of agar containing slugs was excised and placed upside down on a spacer attached to a 35 mm glass bottom dish (12 mm diameter glass, Iwaki). The spacer was filled with liquid paraffin to prevent desiccation during observation and to avoid light scattering. Slugs covered with agar were pushed with a 5 mm diameter plastic rod using a micromanipulator system (MM-94 and MMO-4, Narishige, Japan) (Supplementary Fig. 4a). In the micropipette assay, a piece of agar with slugs was excised and placed directly on a 35 mm glass bottom dish (12 mm diameter glass, Iwaki). A wet paper was placed in the dish and the agar piece was covered with liquid paraffin. A Femtotip microcapillary (1 µm tip diameter, Eppendorf, Germany) was mounted onto a Femtojet pump and micromanipulator (Eppendorf). The slug was pricked with the pipette using manual operation with the manipulator (Supplementary Fig. 4b). During mechanical stimulation, images of slugs expressing GCaMP6s were acquired at 5 s intervals using epifluorescence microscopy.