Slide chemotaxis chamber

The µ-Slide Chemotaxis chamber (ibidi, Martinsried, Germany) was used for quantitative chemotaxis experiments. Developed cells were pipetted into the seeding chamber and then the rest of the chamber was filled with DB (Supplementary Fig. 6A). Then, a 10× solution of the desired cAMP concentration (dissolved in DB) was loaded in the chamber opposite the seeding chamber (Supplementary Fig. 6A). Cells were allowed to settle for 30 min before being imaged every 2 min for 4 h by Phase Contrast microscopy using a ×5 objective (NA 0.16). A detailed description of the microscope is given below. This setup differs from the manufacturer’s recommendation, since cells are loaded into one of the big reservoirs (“seeding chamber”), rather than the narrow central reservoir (“observation area”). This setup is ideal for imaging and tracking of fast migrating cells like Dictyostelium, since cells are allowed to travel the entire distance from one reservoir to the other (1 mm).

6 µL of PC3 cell suspension (3 × 10⁶ cells/mL) were plated in the observation area (3 observation areas/chamber) of a µ-Slide Chemotaxis chamber (ibidi GmbH, Gräfelfing, Germany) using RPMI 10% FBS medium and incubated to allow cell attachment. After 6 h, 65 µL of control medium (EndoGRO basal medium) were added to the right reservoirs of the chamber (3 right reservoirs/chamber). 65 µL of control basal medium or PTEC/HMEC/TRPC3-overexpressing HMEC conditioned medium were added to the left reservoirs of the chamber (3 left reservoirs/chamber). Experiments were performed using the same set-up described for the wound healing assays with a Nikon Plan 10×/0.10 objective. Images of the observations chambers were acquired for 10 h every 10 min using MetaMorph software. Image stacks were analyzed with ImageJ software.

EGFP‐Lifeact ER‐Hoxb8 monocytes were seeded into a narrow channel of a fibronectin coated µ‐slide chemotaxis chamber (Ibidi) which is connected with two reservoirs. After 30 min, the chemotaxis chamber was filled with RPMI 1640 (bicarbonate) medium plus 10% FCS and 1% Pen/Strep. The reservoirs were filled with medium containing 0.003% Patent Blue V (Sigma–Aldrich) (blue dye) either with or without complement C5a (20 nm). The observation area was imaged with an inverted Axio Observer.Z1 epifluorescence microscope (Zeiss) with 10×/0.25 objective. Images were acquired every minute for up to 6 h and analyzed with ImageJ (National Institutes of Health) using a manual tracking plugin and the chemotaxis and migration tool from Ibidi.