Cells were plated at 40,000-50,000 cells ml-1 in 2 ml of media in 35 mm dishes. MDA-MB-231 cells were incubated for 2 hrs supplemented with blebbistatin (Sigma Aldrich), ML-7 (Sigma Aldrich), Y-27632 (Calbiochem, Billerica, MA, USA), P5D2 β1 integrin blocking antibody (mouse mAb, ascites, from Mark Ginsberg, University of California, San Diego) [41 (link)], calyculin A (Santa Cruz Biotechnology, Dallas, TX, USA) and MnCl2 (Fisher) and MTLn3 cells were incubated for 12 hrs with supplemented with calyculin A, MnCl2, Y-27632, and P5D2 on contact guidance cues in imaging media. Substrates with attached cells were inverted onto two strips of double sided tape attached to a microscope slide to generate a flow chamber. The chamber was filled with imaging media and sealed with a 1:1:1 mixture of vasoline, lanolin and paraffin wax. Chambers were imaged by phase contrast microscopy on a heated stage at 37°C every 2 min for 12 hrs. Images were captured at 10× (NA 0.50, Nikon) as described above.
Cell centroids were identified and tracked manually using the MTrackJ plugins of ImageJ (National Institutes of Health, Bethesda, MD, USA). Cell speed and directionality were calculated over a time lag of 2 min averaged over 12 hrs as described in a previous paper [15 ].
Cell centroids were identified and tracked manually using the MTrackJ plugins of ImageJ (National Institutes of Health, Bethesda, MD, USA). Cell speed and directionality were calculated over a time lag of 2 min averaged over 12 hrs as described in a previous paper [15 ].