Information S1 for details of kidney fibroblast isolation. All animal studies were approved by the Institutional Animal Care and Use Committee at Georgia Institute of Technology. Flow Cytometry To determine cell phenotype, cells were analyzed with a LSR-II flow cytometer. Briefly, cells were detached, centrifuged, and separated into 100 ml aliquots then labeled with either 2% PerCP- CD45, PE-Sca1, and APC-CD11b antibodies or PE-CD61, FITC-CD29, and APC-VCAM1. A negative control for each cell type was used to determine positive populations. All studies were performed in triplicate with n = 100,000 events per sample. Conditioned Media Collection Murine mammary carcinoma 4T1 cells were cultured to confluency in 10 cm dishes with standard growth media, then washed in PBS, and incubated in 8 mL unsupplemented DMEM for 24 hours. Cell-free TCM was obtained by centrifugation and filtration. TCM was prepared in a single batch, aliquoted and frozen at 280uC for future use. For all assays with TCM, control media was unsupplemented DMEM. 3D Co-Culture Model To create the 3D ‘sandwich’ model either 4T1 or MSCs were 2 first plated in a 24-well plate at a density of 10,000 cells/cm . Next, 500 ml of a 2 mg/ml collagen solution (pH 7.4) was added to the top of the first cell layer. The collagen solution was prepared by diluting 4.0 mg/ml type I rat tail collagen (BD Bioscience) with serum-free DMEM and buffering solutions. After the collagen was polymerized, fluorescently-labeled MSCs (stained with CM-DiI (Invitrogen) as previously described [57]) were added to the top of 2 the collagen gel (10,000 cells/cm ), and the plate was incubated overnight before imaging with the Nikon Eclipse Ti inverted epifluorescent microscope (Fig. 2G,H). Microrheological Characterization Multiple particle tracking microrheology (MPT) was used to measure the mechanical properties within the cytoplasm of living cells [58]. With this technique, the thermal displacements of fluorescent probe particles are monitored with a fluorescent microscope and related to the viscoelastic properties of the fluid surrounding the tracked particles using the Stokes-Einstein equation [26]. For these studies, cells were cultured to 60–80% confluency on 35 mm glass bottom dishes. Fluorescent 100 nm microspheres, to be used as mechanical probes, were diluted 1:10 in 100% ethanol and dialyzed overnight against 100% ethanol at 4uC. The microsphere solutions were then equilibrated to room temperature, sonicated and briefly centrifuged, to remove particle aggregates. 20 mL of the particle solution was then added to the surface of each macrocarrier. The macrocarriers were dried overnight in the dark and used the next day to inject microspheres into the cell cytoplasm with the PDS- 1000 Biolistic Helium Particle Injection System and 1800 psi rupture discs. After injection, microspheres on the cell surface were removed by extensive washing with PBS, and the cells were incubated overnight in growth media. The following day growth media was replaced with serum-free media, which was used in all experiments. Injected cells were placed on the stage of a Nikon Eclipse Ti inverted epifluorescent microscope, which was maintained at 37uC and 5% carbon dioxide throughout the experiment using an In Vivo Scientific environmental cell chamber. Twenty-second videos of particle diffusion in cells were collected at 33 ms temporal resolution (30 frames per second) with the Photometrics QuantEM Mechanical Changes of MSCs and Fibroblasts CCD camera (Princeton Instruments). The Nikon CFI Apochro- mat TIRF 1006 oil-immersion lens was used for particle tracking. With this lens, which has a high numerical aperature (NA = 1.49) and a reduced signal to noise ratio, it was possible to obtain 5 nm spatial resolution. This value was determined by tracking particles immobilized on a glass coverslip with a strong adhesive. High spatial resolution is obtained by tracking the 2D location of the intensity-weighted centroid of a particle that remains in the focal plane over the 20-second period. A custom MPT routine incorporated in the MetaMorph software (Molecular Devices; Downington, PA) was used to simultaneously monitor the coordinates of 10–20 particles per video. For each condition, particles were tracked in a minimum of 12 cells using 2–3 plates per condition. Particle tracking data was analyzed using a custom routine written for MATLAB software. Briefly, the coordinates of the particle centroids were transformed into families of time-averaged mean squared displacements (MSDs), with MSD defined as ,Dr2(Dt). = ,[x(t+Dt)2x(t)]2+[y(t+Dt)2y(t)]2.. The ensemble- averaged time-dependent MSDs (,,Dr2(Dt)..), reported in Figure 5A–B, were then calculated from the individual particle time-averaged MSDs (,Dr2(Dt).). The mechanical properties of the viscoelastic cytoplasm were then determined from the amplitude, ,,Dr2(v).., and logarithmic slope, a(v) = d ln ,,Dr2(t)../d ln t, of the ensemble-averaged time-dependent MSDs ((,Dr2(Dt).)), where v = 1/t [59]. To facilitate this analysis, the Stokes-Einstein equation was written in the Laplace domain, where the viscoelastic modulus was defined as G(s) = kBT/pas,,Dr2(s).. (kB = Boltzman constant, T = abso- lute temperature, s = Laplace frequency, a = particle radius). The frequency-dependent complex shear modulus (G*(v)) was then determined from the projection of G(s) in the Fourier domain. The real and imaginary components of the complex shear modulus (G*(v)), which are the viscous (G0(v)) and elastic (G9(v)) moduli, respectively, were reported in Figure 5. The phase angle (d, where d = arctan (G0(v))/G9(v)) was used to characterize the degree of stiffness (Fig. 5D), where d = 90u for a viscous liquid, d = 0u for a Hookean solid, 0u,d,90u for a viscoelastic material. Centrifugal Force Adhesion Assay Cells were seeded at 10,000 cells/cm2 in normal growth media and cultured to 80% confluency in a tissue culture treated 96-well plate. Normal growth media was removed and cells were then incubated in serum-free TCM or CM for 24 hours. Cells were then treated with 2 mM Calcein AM (Anaspec), a transmembrane fluorescent viability marker, in PBS+2 mM dextrose for 20 min- utes at 37uC. Cells were then carefully rinsed and covered with PBS-dextrose before an initial emission reading was taken at 485 nm excitation, 535 nm emission on a DTX-800 Multimode Detector microwell plate reader. (Beckman Coulter). Next, sealing tape (Nalge Nunc) was applied to the plate before inverting it and centrifuging it at 600 RPM (64 g) for 5 minutes in a Beckman Coulter Allegra 25R centrifuge (TS-5.1-500 rotor). Wells were then carefully aspirated and rinsed to remove any floating cells, again PBS-dextrose was added and a final emission reading was taken. Adherent fraction was calculated by normalizing the final florescence with the pre-spin values [60]. Immunoflourescence Assays For visualization of cytoskeletal proteins, cells cultured on glass cover slips were briefly extracted in a buffer containing 80 mM PIPES (pH 6.8), 1 mM MgCl2, 5 mM EDTA, and 0.5% Triton X-100 before fixation with 0.5% glutaraldehyde in PBS. The reaction was quenched with 1 mg/mL sodium borohydride, PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33248