Computational protocol: From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

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Protocol publication

[…] After 5 days, gels were mounted on slides, fixed and stained with Carmine . Confocal stacks of the whole mounted gels were acquired using an automated systematic random sampling method (Leica SP5 software). ImageJ plugins, 3D Object counter and 3D ROI manager were used for shape analysis of the epithelial structures. To analyze the shape of the structures, we chose the approach of fitting the structure into an ellipsoid. The shape descriptors Elon1 and RatioVolEllipsoid were chosen to analyze the types of structures formed: acini or ducts . Briefly, Elon1 is the ratio between the major axis and the middle axis of the 3D fitted ellipsoid and RatioVolEllipsoid is the ratio between the volume of the 3D structure and the 3D fitted ellipsoid. Structures smaller than 300 voxels were excluded from the analysis. In general, for structures that are elongated and not branching, the corresponding ellipsoid has a major axis far larger than the middle axis, and so Elon1 is substantially greater than 1. If the structure is spherical as in the case of an acinus, Elon1 is approximately 1. However, a branching duct does not fit well into an ellipsoid, and this can result in low or intermediate Elon1 values. In order to resolve this, we used the ratio r of the volume of the ellipsoid by the volume of the structure (1/RatioVolEllipsoid). Non-branching ducts have a high Elon1 and low r, while branching ducts have a low or intermediate Elon1 and high r. Contrariwise, acini have low Elon1 and low r. In order to distinguish acini and ducts we defined a “shape score” as log(Elon1*r). We computed the normalized histogram of the diverse shape scores for epithelial structures ranging from acini to ducts. [...] Evaluation of viscoelastic properties was performed using a Kinexus Pro Rheometer (Malvern, UK). Using Parallel-plate geometry (20 mm×65 mm), gels were placed onto the lower plate of the rheometer, while the higher plate oscillated on the sample at a gap size of 1 mm. For each of the samples, small deformation linearity (linear viscoelastic region) was identified via strain measurement under the application of controlled stress sweeps. Optimum conditions such as shear strain at 1.0% of the angular single frequency (1 Hz) were selected. All measurements were performed at a temperature of 37±0.02°C (n = 4, from two independent experiments). Shear modulus (G′) measurements were derived from G′/G″-frequency (ω) plots produced by single frequency measure. Data was analyzed using proprietary software (rSpace). […]

Pipeline specifications

Software tools ImageJ, RSpace
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens