Dictyostelium discoideum Cell Growth Dynamics

AX2 axenic cells of Dictyostelium discoideum were grown in HL5 medium with glucose (Formedium, Norfolk, UK) at 22 °C in tubes with shaking conditions (180 rpm for oxygenation). Exponentially growing cells were harvested, counted and deposited in 6-well plates, with a initial surface cell density ρ₀ between 5 × 10³ and 2 × 10⁵ cells/cm². Cells were submerged below liquid growth medium with height ranging from h = 0.5  to 3 mm. The temperature was kept constant at 22 °C.
We observed the growth and aggregation of cells under this free liquid film for days. We also performed experiments where the liquid film is topped with a thin layer of oil (low viscosity paraffin oil, ref. 294365H, VWR Chemicals). Because the product of solubility and diffusion coefficient of oxygen in oil is high, the presence of oil does not appreciably reduce the availability of oxygen and the final mean aggregate size is close (within 10%) to that observed without oil.
The growth and aggregation of Dd cells were observed in transmission with three types of microscope: (i) a TE2000-E inverted microscope (Nikon) controlled with Micromanager (version 2.0 gamma) and equipped with a motorized stage, a 4X Plan Fluor objective lens (Nikon) and a Zyla camera (Andor) using brightfield for most of the timelapse experiments lasting up to several days (Figs. 1a–c, 2b, c, Supplementary Figs. 4–5, and Supplementary Movies 1, 3, and 5), (ii) a binocular MZ16 (Leica) controlled with LAS X software (version 3.4.2 Leica) and equipped with a TL3000 Ergo transmitted light base (Leica) operated in the one-sided darkfield illumination mode and a Leica LC/DMC camera (Leica) for large field experiments (Fig. 1e, Supplementary Figs. 1–2 and Supplementary Movie 4) and finally (iii) a confocal microscope (Leica SP5) controlled with the LAS software (version 3.4 Leica) and equipped with a 10X objective lens for larger magnification experiments (Supplementary Fig. 3A–C and Supplementary Movie 2) and with a Tile Scan to create large field reconstituted mosaic images (Fig. 2a).