Sup_inf_protocol_f3.doc - Supporting Information 4D...

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Supporting Information 4D traction force microscopy reveals asymmetric cortical forces in migrating Dictyostelium cells Delanoë-Ayari, H. and Rieu, JP Université de Lyon, F-69000, France; Univ. Lyon 1, Laboratoire PMCN; CNRS, UMR 5586; F-69622 Villeurbanne Cedex, France Sano, M. Department of Physics, University of Tokyo, Tokyo 113-0033, Japan In this supplement, we provide a detailed Materials and Methods section, two tests of the reconstruction method, and we present the 3D displacement map of a fibroblast cell. 1. Materials and Methods Cell culture and developmental conditions All experiments were conducted with AX-2 wild-type Dictyostelium discoideum cells at 21°C in the vegetative state. Cells were cultivated in petri dishes (88 mm diameter) to densities of ~5×10 6 cells/ml in HL5 media. They were washed twice in Phosphate Development Buffer (PDB: KH 2 PO 4 , 5mM; Na 2 HPO 4 -12H 2 O, 5mM; 2mMMgSO 4 2mM; CaCl 2 0.2mM; pH 6.9) and a droplet of 200 l at 5 x10 4 cells/ml in growth medium was deposited on the 22 mm circular elastomer layer. Polyacrylamide Gels Flexible polyacrylamide gel sheets were prepared and calibrated, as described previously [1], with 2.2% acrylamide, 0.25% bis, ammonium persulfate (10% w/v solution, 1:125 volume), TEMED (1:1250 volume; all Bio-Rad products) and 0.22% v/v of 0.2-μm fluorescent beads (2% solid green
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beads, Molecular Probes). Their elastic modulus was measured as previously described [1] at E=400 100 Pa while the Poisson ratio was always taken as 0.5 [2, 3]. Cell speed Cell speed is calculated as the 2D cell mass center displacement during 8.5 sec. For this short timescale, it is a measurement of the cell protrusion activity. Detection of 4D substrate deformations We used a confocal microscope (Leica TCS-SP5) to detect bead displacements in xyzt dimensions (4D). We recorded simultaneously the cell shape at about +1 m from the elastomer surface (transmission channel) and a stack of fluorescence images every 0.35µm between z= 0 and z= - 6 m. In a continuous recording mode, at (128x128) pixels image size, the time lapse time step between two stacks was 8.5 sec, and xyz time series were recorded during about 7 min. At the end of that period, the undisturbed bead reference stack could be recorded, either because cells escaped the recorded field of view, or because cells were gently detached by adding a buffer droplet above the sample. Stacks where aligned at the pixel resolution in the x,y and z direction using image correlations with Matlab software. To measure the three dimensional bead displacements from the reference stack , we used the 3D tracking code created by John C. Crocker and Eric R. Weeks using the feature3D algorithm on IDL software [4]. To obtain a high 3D spatial resolution, we used a high density of beads. Because the bead displacement was quite greater than the bead to bead average distance we have first to calculate the rough displacements in the xy and z plane and correct for it before applying the tracking 3D algorithms.
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  • Spring '13
  • L
  • U. S. Schwarz, J. P. Rieu, A. Rusanov, M. Sano, B. Sabass

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