3d slide chemotaxis devices

For chemotaxis of growth phase cells versus pterines, a final concentration of 100 µM folate (Sigma-Aldrich; F8758) was used to set up the gradient in PDMS microfabricated or 3D µ-slide chemotaxis devices (ibidi GmbH, Gräfelfing, Germany) with a VitroGel Hydrogel matrix (TheWell Bioscience Inc., North Brunswick, NJ, USA). For chemotaxis experiments with cells during the aggregation competent stage, cAMP (Sigma-Aldrich; A9501) was used at a final concentration of 10 µM in micropipette assays, PDMS microfabricated devices, and 3D µ-slide chemotaxis devices (ibidi GmbH, Gräfelfing, Germany) with VitroGel Hydrogel (TheWell Bioscience Inc., North Brunswick, NJ, USA) or rat collagen type I (ibidi GmbH, Gräfelfing, Germany) matrices.

To analyze the position of the centrosome in relation to the nucleus during migration in 3D environments, 3D µ-slide chemotaxis devices (ibidi GmbH, Gräfelfing, Germany) were used in combination either with VitroGel Hydrogel (TheWell Bioscience Inc., North Brunswick, NJ, USA) or rat collagen type I (ibidi GmbH, Gräfelfing, Germany) matrices.
Hydrogel: 100,000 Dictyostelium cells of the developmental phase were suspended in PB and mixed with hydrogel (v/v 1:2), loaded into a 3D µ-slide chemotaxis chamber (6 µL into the middle channel), and were allowed to settle down for 1 h at RT. To set up a gradient of cAMP, a final concentration of 10 µM cAMP was loaded (65 µL) into one outer compartment of the chamber, the other one was filled with PB (65 µL).
Collagen: 100,000 Dictyostelium cells of the developmental stage were suspended in a final volume of 75 µL containing rat tail collagen type I (ibidi GmbH, Gräfelfing, Germany; 1.5 mg/mL) in PB supplemented with 6.7 mM NaOH, 1.2 mM CaCl₂, and 0.2% NaHCO₃. The cell–collagen mix was loaded into the 3D µ-slide chemotaxis chamber (6 µL into the middle channel), and was allowed to settle down for 30 min at RT for polymerization [58 (link)]. Then, 100 nM cAMP was added to one side (65 µL) to generate a chemoattractant gradient, and the other side was filled with PB (65 µL).