Chemotaxis tool

Chemotaxis was analyzed with neutrophils resuspended in HBSS supplemented with 15mM HEPES (pH 7.4) and 0.05% fatty acid and endotoxin-free BSA. For integrin-dependent chemotaxis, neutrophil migration on a glass bridge was monitored by time lapse-imaging for 30 minutes in Dunn chambers (Hawksley, Lancing, UK). Dunn chambers were assembled as previously described (21 (link)) with 300nM fMLF as the chemoattractant. For integrin-independent chemotaxis, neutrophils were mixed with a 3D collagen matrix (A1048301, Roche Diagnostics, Mannheim, Germany), which was prepared as per manufacturer’s instructions, and left to polymerize in a humidified incubator at 37°C at 5% CO₂ before cells were allowed to migrate towards 300nM fMLF in Chemotaxis μ-slides (Ibidi, Martinsried, Germany). Images were acquired on a Leica IRB inverted microscope with temperature-controlled chamber, automated stage (Prior, Cambridge UK), Orca camera (Hamamatsu, Welwyn Garden City, UK) and Micromanager image acquisition software (Fiji). Paths of individual cells were tracked using the manual tracking plug-in into Image J and tracks analyzed using the Chemotaxis Tool (Ibidi) plug-in into Image J as described (19 (link)).

For chemotaxis assays, neutrophils were resuspended in HBSS supplemented with 15 mM HEPES pH 7.4 and 0.05% fatty acid and endotoxin-free BSA. Dunn chambers were assembled as described (21 (link)), and chemotaxis assays were carried out with MIP2 (R&D Systems) as chemoattractant. Cells were monitored by time-lapse imaging for 30 min using an inverted RMDIB microscope (Leica) equipped with temperature-controlled chamber, automated stage (Prior), Orca camera (Hamamatsu), and Micromanager image acquisition software (Fiji). Paths of all individual neutrophils were tracked using the manual tracking plug-in into ImageJ. Tracks were subsequently analyzed using the Chemotaxis Tool (Ibidi) plug-in into Image J.

Murine NIH 3T3 fibroblasts were plated on collagen type I-coated μ-slides (IBIDI USA Inc., Madison, WI) and incubated for 4 hours at 37 °C in 5 % CO₂/95 % air in complete DMEM media supplemented with 10 % FBS to insure complete cell adhesion on a bridge. Subsequently, rhCTHRC1 protein (Sino Biological Inc., Beijing, China) was added at 1000 ng/ml, and imaging has been performed for 14 hours in a serum-containing medium at 37 °C using time-lapse phase contrast microscopy with a Cell Observer microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Recorded stacks of images were transferred to ImageJ [19 (link)] and further analyzed with the MTrackJ plugin and Chemotaxis Tool (IBIDI USA Inc., Madison, WI, USA). The trajectories of individual cells were evaluated for accumulated distance migrated (Ad) and for cell velocity. Directness was calculated as a ratio of Euclidean distance (Ed) to the accumulated distance (Ed/Ad) (Fig. 6). Additionally, elongated morphology cell shape was studied (horizontal polarization), which was defined as maximal cell dimension using linear approximation. In preliminary experiments, we found that pro-motile and polarization effects of the CTHRC1 treatment were pronounced in the time window from 4–12 hours, and thereafter all calculations were performed for this time period.

Individual neural crest cells located at the edge of explants were tracked using Manual Tracking plug-in in FIJI. Tracks were analysed using Chemotaxis Tool plug-in from IBIDI to retrieve individual cell’s speed.