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C. Ballistic Injection of Nanoparticles

A biolistic particle delivery system (PDS-1000/He system, Cat. No. 165-2258) equipped with a hepta adaptor (Cat. No. 165-2225) from Bio-Rad (Hercules, California) is used to inject nanoparticles into live cells. Biolistic machine's accessories include 2200 psi rupture disks (Cat. No. 165-2334), macrocarriers (Cat. No. 165-2335), and stopping screens (Cat. No. 165-2336), all from Bio-Rad. Dry vacuum pump (model 2560, Cat. No. 2560C-02) is from Welch Rietschle Thomas (Stokie, Illinois). Rupture disks are pretreated with isopropanol (Cat. No. I9516; Sigma) prior to ballistic injection.

D. Encapsulation of Cells in a 3D Matrix

Puramatrix (Cat. No. 354250) from BD Biosciences (San Jose, California) is used as 3D matrix to study cell mechanics in 3D. Encapsulation is done in an 8-well, chamber glass slides (Cat. No. 155411; Lab-Tek). Sucrose (Cat. No. 407205) used during encapsulation is from JT Baker (Phillipsburg, New Jersey).

E. Imaging of Fluctuating Nanoparticles Embedded in the Cell

BIN experiments are conducted with a Nikon TE2000-E inverted microscope equipped for epifluorescence, using a Nikon PlanFluor 60 x oil immersion lens (NA 1.3). Movies of fluctuating fluorescent nanoparticles are captured onto the random access memory of a PC computer via a Cascade 1K camera (Roper Scientific, Tucson, Arizona) controlled by the Metavue software (Universal Imaging Corp., Sunnyvale, California), at a frame rate of 30 Hz. Time-dependent coordinates of the centroids of fluorescent nanoparticles are obtained using particle-tracking routines built into the Metamorph Imaging Suite (Universal Imaging Corp.).

III. Procedures

A. Preparation of Nanoparticles

Nanoparticles are coated onto macrocarriers prior to ballistic injection. To accomplish this, they are spread, then left to dry onto the macrocarrier. Since polystyrene nanoparticles are obtained as 2% (w/v) aqueous suspension, they are dialyzed against 100% ethanol prior to coating to accelerate the drying process.

in the cytoplasm of cells embedded in the matrix. (B) Mean cellular creep compliance (cytoplasmic deformability) and (C) mean frequency-dependent viscous and elastic moduli, G (o) (circles) and G" (o) (squares), of HUVECs embedded in a 3D matrix. Figures reprinted with permission of Panorchan et al. (2006) and the Biophysical Society.

1. Preparation of Carboxylated Nanoparticles a. Steps i. Pipette 3 ml of stock nanoparticles into a 6-cm piece of dialysis tubing sealed at one end and prewet with ethanol.

ii. Dialyze against 3 liters of ethanol with gentle stirring at 4 °C for 8 h. Repeat three times with fresh ethanol.

iii. Aliquot the nanoparticle solution into tight, sterile 1.5-ml Eppendorf tubes, and store at 4 °C.

B. Ballistic Injection of Nanoparticles

A commercial biolistic particle delivery system (Bio-Rad) is used to deliver 100-nm diameter polystyrene fluorescent nanoparticles to culture cells. Helium gas at 2200 psi is used to force a macrocarrier disk coated with fluorescent nanoparticles to collide into a stopping screen. The forces of collision cause the nanoparticles to dissociate from the macrocarrier and bombard target cells.

Adherent cells are plated on 100-mm tissue culture dishes and grown to ^90% confluency. Prior to injection, the medium is aspirated and the dish is loaded onto the machine. Once ballistically injected, the cells are washed extensively and allowed to recover for 1 h, before replating onto 35-mm glass bottom dishes overnight for particle-tracking analysis.

1. Coating of Macrocarriers a. Materials i. Particle suspension prepared in step (Section III.A. 1.a.iii)

ii. Macrocarriers b. Steps i. Add a 17-ml drop of nanoparticle suspension onto a macrocarrier. Prepare seven macrocarriers per ballistic injection.

ii. Holding a pipetter with a 20-ml pipette tip sideways, spread the drop of solution on the macrocarrier evenly with a brushing motion. Coat all seven macrocarriers. Wait 5 min and apply a second coating.

iii. Let stand for 30 min to ensure complete dryness before ballistic injection.

2. Ballistic Injection of Fluorescent Nanoparticles a. Materials i. Dried nanoparticles on macrocarrier prepared in step (Section III.B.1.b.iii)

ii. Isopropanol iii. HBSS

iv. HUVEC's complete growth medium (F-12K medium, 10% FBS, 0.1-mg/ml heparin, 0.05-mg/ml ECGS, 1% Pen-Strep solution)

b. Steps i. Autoclave hepta adaptor, stopping screens, and macrocarrier launch assembly.

ii. Soak the rupture disk in isopropanol for 2 sec and place a rupture disk in the round slot inside the hepta adaptor.

iii. Screw the hepta adaptor containing the rupture disk onto the helium gas acceleration tube inside the ballistic chamber. Insert torque wrench (provided with the machine) in the small upper ring of the hepta adapter assembly and tighten the hepta adaptor.

iv. Place the coated macrocarriers into seven-hole macrocarrier holder with the coated side down (particles toward the sample). Use the seating tool (provided with the machine) to fit macrocarriers firmly into the macrocarrier holder.

v. To eliminate aggregates of fluorescent nanoparticles on the macrocarrier (problematic for particle-tracking analysis), gently rub the macrocarrier with a clean finger or a small sterile scraper until no distinct pellets of nanoparticles is visible. Aggregates in cells can be distinguished by their anomalously high intensity relative to single beads.

vi. Enclose the macrocarrier holder with stopping screen and stopping screen holder. Place this assembly into the hepta macrocarrier shelf and slide the final assembly into the highest shelf slot position in the ballistic chamber. Align the seven pressure outlets of the hepta adapter with the centers of the seven holes in the macrocarrier holder.

vii. Aspirate medium from previously prepared 100-mm tissue culture dish. Place the tissue culture dish onto the target shelf, and slide it into the slot immediately below the hepta macrocarrier shelf.

viii. Lock the ballistic chamber and pull vacuum to ~28-in. Hg. Hold the pressure and fire until the rupture disk breaks. Release pressure and retrieve tissue culture dish.

ix. Immediately wash cells three times with HBSS and replace medium with HUVEC complete growth medium. Allow cells to incubate for 1 h before replating.

C. Cell Seeding and Encapsulation in a 3D Matrix

Once ballistically injected, cells are now ready for culture. Cells can be either seeded on the surface or encapsulated within Puramatrix peptide hydrogels.

1. Seeding HUVECs on a 2D Surface of Matrix a. Materials i. Puramatrix peptide hydrogel (1% stock)

ii. HUVEC's complete growth medium (F-12K medium, 10% FBS, 0.1-mg/ml heparin, 0.05-mg/ml ECGS, 1% Pen-Strep solution)

b. Steps i. Sonicate hydrogel for 30 min in a bath sonicator.

ii. Dilute the sonicated hydrogel 1:1 with sterile water. Add 100 ml of diluted hydrogel to an 8-well chamber slide (100 ml per well). Use the pipette tip to gently spread the hydrogel solution to cover the entire bottom of each well.

iii. With a pipette tip touching the wall of the well, gently add 300 ml of culture medium to initiate assembly of the hydrogel.

iv. Allow 1 h for gel assembly at room temperature. Change medium every 15 min within the hour.

v. Typsinize and spin down cells at 2000 rpm for 5 min. Collect the pellet and resuspend at 1 x 105 cells/ml. Add 50 ml of the cell suspension to each well. Gently mix the solution with the pipette.

vi. Allow 6-12 h incubation prior to BIN analysis.

2. Encapsulation of HUVECs Within a 3D Matrix a. Materials i. Puramatrix peptide hydrogel (1%, stock)

ii. 10% sucrose solution (use sterile water)

iii. HUVEC's complete growth medium (F-12K medium, 10% FBS, 0.1-mg/ml heparin, 0.05-mg/ml ECGS, 1% Pen-Strep solution)

b. Steps i. Sonicate hydrogel for 30 min in a bath sonicator.

ii. Typsinize and spin down cells at 2000 rpm for 5 min. Remove the supernatant and add 5 ml of 10% sucrose as wash solution. Resuspend cells and repellet, then remove the wash solution. Resuspend cells at 1 x 106 cells/ml with fresh 10% sucrose solution.

iii. Mix the sonicated hydrogel with cell suspension at 1:1 ratio. Add 200 ml of the mixture to each well in an 8-well chamber slide. With a pipette tip touching the wall of the well, gently add 200 ml of culture medium to initiate assembly of the hydrogel. It is important to proceed as quickly as possible, as the hydrogel solution has a very low pH, which is harmful to the cells.

iv. Allow 1 h for gel assembly at room temperature. Change medium every 10 min within the hour. Incubate overnight prior to BIN analysis.

D. BIN Analysis

BIN analysis involves capturing motions of embedded nanoparticles with time lapsed movies. These movies of trapped nanoparticles inside a cell are then analyzed by a custom particle-tracking routine incorporated into the Metamorph imaging suite as described (Tseng and Wirtz, 2001). Individual time-averaged MSDs, (Ar2(t)) = (\x(t +t) - x(t)]2 + [y(t +t) - y(t)]2), where t is the timescale, are calculated from the 2D trajectories of the centroids of the nanoparticles. With ballistic injection, a large number of cells are available for data acquisition (relative to conventional single cell studies). Typically, a sample size of 30 cells, each containing ~10 nanoparticles, is used per condition. All measurements are performed in an incubator mounted on an inverted microscope maintained at 37 °C with 5% CO2 and humidity. To correlate particle-tracking measurements with cellular structures, the cultures are fixed after measurements, for subsequent labeling with fluorescent antibodies (Fig. 4A).

1. Acquisition of Movies of Fluctuating Nanoparticles a. Steps i. Place previously prepared cell culture chamber on the stage of an epifluores-cence microscope equipped with a live cell incubator maintained at 37 °C and 5% CO2.

ii. Identify ballistically injected cells with a 60 x Plan Fluor objective, using a combination of fluorescence and bright field illumination.

iii. Center on the cell of interest. Adjust the focus under fluorescence illumination to obtain the clearest image of nanoparticles and acquire 20-100 sec of streaming video with a Cascade 1K camera at a frame rate of 30 Hz. To achieve this frame rate, image acquisition region was limited to 500 x 300 pixels, with 3 x 3 binning. Adjustment may be necessary for different cameras. Acquire also a still fluorescence image of the nanoparticles after recording the movie.

iv. Switch to high-resolution bright field and acquire still image of the cell. Save the movie of fluorescence nanoparticles as a stk file and the still images as tiff files.

2. Analysis of Movies a. Steps i. Open an stk movie file of fluctuating nanoparticles in the Metamorph image analysis software. Calibrate pixel distances using a previously acquired image of a stage micrometer. Draw a region of interest, zoom 400%, and duplicate the entire movie sequence with zoom.

ii. Using the "track objects'' command, create inner and outer regions around each nanoparticle in the first frame of the sequence. Do not create regions around aggregated nanoparticles, since these nanoparticles violate assumptions made in our constitutive viscoelastic equations and cannot be used for analysis.

iii. For each nanoparticle, adjust the inner region so that it extends just beyond the edge of the nanoparticle. Adjust the outer region so that it is large enough to encompass the nanoparticle in all the subsequent frames. Duplicate an image of the initial frame with labeled regions visible and save as a tiff file.

iv. Open ''log to excel.'' Track the nanoparticles and log the ID number, frame number, and the time-dependent coordinates, [x(t), y(t)], for each frame. Save this data as an excel spreadsheet.

3. Calculations of MSDs, Creep Compliance, and Viscoelastic Moduli

From the time-dependent coordinates, [x(t), y(t)], the projections of the MSD for each nanoparticle in the x and y directions are calculated using the following formulas (Qian et al., 1991):

Hence, the MSD of the nanoparticle, (Ar2(t)), is simply

The MSD of each probe nanosphere is directly related to the local creep compliance of the cytoplasm, r(x), as (Xu et al., 1998c)

where kB is Boltzmann's constant, T is the absolute temperature of the cell (in Kelvin), and a is the radius of the nanoparticle. Fluctuations due to active motors have also been considered (Lau et al., 2003). The creep compliance (expressed in units of cm2/dyn, the inverse of a pressure or modulus) describes the local deformation of the cytoplasm as a function of time created by the thermally excited displacements of the nanoparticles. The method to obtain frequency-dependent viscoelastic moduli has been described in details in Kole et al. (2004b). Briefly, the complex viscoelastic modulus, G*(m), is obtained using the following equation:

where m = 1/t and 3u{(Ar2(t))} is the Fourier transform of (Ar2(t)). The elastic modulus is the real part of Eq. (4) and the viscous modulus is the imaginary part (Chapter 1 by Janmey et al. and Chapter 2 by Kandow et al., this volume). While G*(m) may be calculated numerically, an analytical solution was obtained by Mason et al. (1997) by approximating the Fourier transformation of (Ar2(t)) using a wedge assumption, which expands (Ar2(t)) locally around the frequency of interest m using a power law and retains the leading term (Mason et al., 1997). The Fourier transform of (Ar2(t)) then becomes:

where a(o) = d ln(Ar2(t))/d lnt|t=1=o is the local logarithmic slope of (Ar2(t)) at the frequency of interest o = 1/t and r is the gamma function. The frequency-dependent elastic and viscous moduli, G and G"', can then be calculated algebraically using the following relationships:

where

2kBT

IV. Pearls and Pitfalls

A. During data acquisition, the bright intensity of large aggregates in the sample will overwhelm the intensity of an individual particle. To circumvent this issue, there are several things to try. First, sonicate the particle suspension for 30 min prior to coating. Second, reduce the volume of particle suspension used for coating the macrocarrier. The particle suspension in ethanol usually becomes more concentrated after several cycles of usage/storage due to evaporation of ethanol, which may be compensated by using a smaller volume. Third, spread the particle suspension on the macrocarrier for longer to avoid visible collection of particles. Finally, while loading a coated macrocarrier onto the macrocarrier holder, be sure to thoroughly rub the macrocarrier with your finger or a scraper.

B. In case of low particle penetration efficiency, ensure while coating the macrocarrier, that the particles are suspended well in the solution. Once dialyzed in ethanol, particles tend to settle quickly to the bottom of the Eppendorf tube. Make sure to mix by pipetting up and down prior to coating every macrocarrier. In addition, ensure that the medium in the tissue culture is removed as much possible during aspiration. Wet layer on the tissue culture plate impedes particle penetration.

C. Maintain high confluence of culture prior to ballistic injection. Lower con-fluency lead to inefficient particle penetration and lower cell viability.

D. One of the most critical steps in ballistic injection is washing. Cells must be wash immediately and vigorously after ballistic injection to remove any lodged particles on the surface of the cells as well as free-floating particles. These particles may be endocytosed by the cells and will appear as extremely fast moving particles under BIN analysis since they move via active transport.

V. Concluding Remarks

A. Unique Advantages of BIN

BIN offers unique advantages compared to conventional particle-tracking nanorheology:

1. In a single ballistic injection, the number of injected cells amenable to measurements increases by a 1000-fold compare to the microinjection technique (Lee et al., 2006; Panorchan et al., 2006).

2. Microinjection of cells is highly inconsistent due to invasive nature of injection and the mechanical trauma to the cells that ensues. With ballistic injection, every cell is injected similarly thus decreasing cell-to-cell variations in the measurements sometimes found following microinjection.

3. With a large population of injected cells, BIN allows us to probe single-cell mechanics in complex geometries and in more physiological situations, including 3D model tissues (Panorchan et al., 2006), cells subjected to shear flows (Lee et al., 2006), and cancer cells at the rear and the edge of a wound (J. S. H. Lee, P. Panorchan, and D. Wirtz, unpublished data).

4. With a large sample size per condition (number of probed cells ^30), our results become much more precise and significant. The small sample size (number of probed cells ~5) of microinjection leads to cell-to-cell variations and potential random experimental errors. BIN provides a more precise and consistent values for global and local viscoelastic properties.

B. Advantages of Traditional Particle-Tracking Nanorheology Are Maintained by BIN

BIN also preserves all the advantages of particle-tracking nanorheology:

1. BIN can measure directly mechanical properties of the cytoplasm (Tseng et al., 2004b). Most current single-cell mechanics methods rely on contact between the cell surface and a physical probe. Therefore, these methods cannot distinguish the contribution of the plasma membrane from those of the nucleus, cytoskeleton, and other organelles without making drastic assumptions. In contrast, BIN directly measures the mechanical properties of the cytoplasm.

2. BIN measures shear rate-dependent viscoelastic moduli. This is particularly crucial for the cytoskeleton, which behaves like a liquid at long timescales (or low rates of shear) and like an elastic solid at short timescales (or high rates of shear).

3. By tracking multiple nanoparticles simultaneously, BIN can measure simultaneously micromechanical responses to stimuli in various parts of the cell. By using video-based multiple particle tracking instead of laser deflection particle tracking (Mason et al., 1997; Yamada et al., 2000), hundreds of nanoparticles embedded in the body of cells can be tracked at the same time.

4. BIN rheological measurements are absolute and compare favorably with traditional rheometric measurements on standard fluids of known viscosity and elasticity (Apgar eta/.,2000; Mason etal., 1997; Xu etal., 1998a,c). This is not the case of some single-cell approaches that rely on the contact between the cell surface and the probe. It is now clear that the apparent viscoelastic moduli measured by magnetocytometry and AFM depend greatly on the type of ligands coated on the magnetic beads or AFM cantilever. Extracellular ligands—including fibronectin, RGD peptide, and ICAM1—coated on magnetic beads and AFM cantilevers lead to vastly different values of (apparent) cell stiffness. Therefore, the measurements of viscoelastic properties of standard materials using these methods cannot be compared to those obtained using a cone-and-plate rheometer.

5. BIN measures both elasticity and viscosity, while many other approaches cannot distinguish the elastic from the viscous responses of the cell directly.

Acknowledgments

This work was partially supported by an NIH grant (R01 GM075305-01) and a NASA grant (NAG9-1563). J.S.H. Lee was supported by a NASA graduate training grant (NNG04G054H). This work was also supported by a graduate training grant from the Howard Hughes Medical Institution.

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