Chemotactic assays were performed as described previously (Suraneni et al., 2012 (link)), with several modifications. Briefly, ARPC3+/+ or ARPC3−/− fibroblast cells were trypsinized, diluted to ∼2 × 106 cells/ml, and plated in a μ-Slide Chemotaxis slides (Ibidi, Martinsried, Germany) precoated with 5 μg/ml of fibronectin and allowed to recover for 5–6 h. The medium was replaced with low-serum medium (DMEM with 0.5% FBS) overnight, followed by replacement with fibroblast medium. Then one of the ports was filled with chemoattractant (500 ng/ml PDGF or EGF) solution. Cell migration in response to chemotactic signal was recorded by placing the μ-Slide on an inverted Nikon Eclipse TE2000-E microscope with a 37°C incubator and 5% CO2 for a period of 12 h with frames taken every 20 min. Cell-trajectory analysis was performed as described previously (Suraneni et al., 2012 (link)).
For the mixed-cells experiment, wt and mutant fibroblasts were labeled with live-cell tracker green CMFDA (C7025; Life Technologies, Grand Island, NY) and red CMTPX (C34552; Life Technologies), respectively, before trypsinization and mixing. The ARPC3+/+ cells with ARPC3−/− cells conditional medium experiments were performed by collecting the medium from ARPC3−/− cell dishes after 12 h of culturing. The remaining procedure was performed as described in the preceding section.
For the mixed-cells experiment, wt and mutant fibroblasts were labeled with live-cell tracker green CMFDA (C7025; Life Technologies, Grand Island, NY) and red CMTPX (C34552; Life Technologies), respectively, before trypsinization and mixing. The ARPC3+/+ cells with ARPC3−/− cells conditional medium experiments were performed by collecting the medium from ARPC3−/− cell dishes after 12 h of culturing. The remaining procedure was performed as described in the preceding section.
