The authors would like to acknowledge Lloyd Ung for constructive input in early versions of this technique, Dr. Jeremy Agresti for pressure cap design tips, and Dr. Dongping Qi for his help in fabricating the pressure cap. We are grateful to the laboratories of M. Teitell and P. Gunaratne for providing a variety of cell samples for testing. We are grateful to the National Science Foundation (CAREER Award DBI-1254185), the UCLA Jonsson Comprehensive Cancer Center, and the UCLA Clinical and Translational Science Institute for supporting this work.

1. Microfluidic Device Design
NOTE: The device design has four basic functional regions: entry port, cell filter, constriction array, and exit port (Figure 1). The overall design can be applied to a wide array of cell types, with minor adjustments to dimensions. Provided here are a few basic design recommendations along with device parameters that are effective for a selection of both primary and immortalized cells.
<ol>
	<li>Select the width of constriction array channels (...

<ol>
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The authors have no conflicts of interest to disclose.

Here we provide a comprehensive experimental procedure for analyzing the deformation of cells transiting through constricted microfluidic channels using pressure-driven flow. A MATLAB script enables automated data processing (Supplemental Material); an updated version of the code is maintained (<a href="http://www.ibp.ucla.edu/research/rowat" target="_blank">www.ibp.ucla.edu/research/rowat</a>). More broadly, the techniques presented here can be adapted in many cell-based microfluidic assays, including the effect of cyto...

Changes in cell shape are critical in numerous biological contexts. For example, erythrocytes and leukocytes deform through capillaries that are smaller than their own diameter1. In metastasis, cancer cells must deform through narrow interstitial gaps as well as tortuous vasculature and lymphatic networks to seed at secondary sites2. To probe the physical behavior of individual cells, microfluidic devices present an ideal platform that can be customized to study a range of cell behaviors including their ability to migrate through narrow gaps3 and to passively deform through micron-scale constrictions3-9. Polydimethylsiloxane (PDMS) microfluidic devices are optically transparent, enabling cell deformations to be visualized using light microscopy and analyzed using basic image processing tools. Moreover, arrays of constrictions can be precisely defined, enabling analysis of multiple cells simultaneously with a throughput that exceeds many existing techniques10,11.
Here we present a detailed experimental protocol for probing cell deformability using the &#8216;Cell Deformer&#8217; PDMS microfluidic device. The device is designed so that cells passage through sequential constrictions; this geometry is common in physiological contexts, such as the pulmonary capillary bed12. To gauge cell deformability, transit time provides a convenient metric that is easily measured as the time required for an individual cell to transit through a single constriction4,6. To maintain a constant pressure drop across the constricted channels during cell transit, we use pressure-driven flow. Our protocol includes detailed instructions on device design and fabrication, device operation by pressure-driven flow, preparation and imaging of cells, as well as image processing to measure the time for cells to deform through a series of constrictions. We include both device designs and vision data processing code as supplemental files. As a representative sample of data, we show cell transit time through a series of constrictions as a function of the number of constrictions passaged. Analysis of the timescale for cells to transit though narrow constrictions of a microfluidic device can reveal differences in the deformability of a variety of cell types4,5,13. The device demonstrated here uniquely surveys cell transit through a series of micron-scale constrictions; this design emulates the tortuous path that cells experience in circulation and also enables probing additional physical characteristics of the cells such as relaxation time.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pluronic F-127 Block Copolymer Surfactant&#160;</td>
			<td style="width:71px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">8409400</td>
			<td style="width:167px;">Produced by BASF, also available through Sigma</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">PDMS base and crosslinker</td>
			<td style="width:71px;">Essex Brownell</td>
			<td style="width:119px;">DC-184-1.1</td>
			<td style="width:167px;">Product commonly named Sylgard 184 Elastomer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Oxygen plasma discharge unit</td>
			<td style="width:71px;">Enercon</td>
			<td style="width:119px;">Dyne-A-Mite 3D Treater</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Biopsy Punch, Harris Uni-Core (0.75 mm)</td>
			<td style="width:71px;">Ted Pella, Inc.</td>
			<td style="width:119px;">15072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight Ferrule, 1/32&#34;</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-113</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight III Fitting, 10-32</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-300X</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyetheretherketone (PEEK) tubing, outer diameter = 1/32&#34;or 0.79 mm</td>
			<td style="width:71px;">Valco</td>
			<td style="width:119px;">TPK.515-25M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyethylene (PE-20) tubing, 0.043&#34; or 1.09 mm</td>
			<td style="width:71px;">Becton Dickinson</td>
			<td style="width:119px;">427406</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pressure regulator</td>
			<td style="width:71px;">Airgas or Praxair</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Polyurethane tubing, 5/32&#8221; OD</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5648K284</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Push-to-connect fittings</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5111K91</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Voltage to Pressure (E/P) Electropneumatic Converter</td>
			<td style="width:71px;">Omega</td>
			<td style="width:119px;">IP413-020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">16-bit,250 kS/S, 80 Analog Inputs Multifunction DAQ</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">NI PCI 6225-779295-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Analog Connector Block-Screw Terminal</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SCB-68-776844-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">LabView System Design Software</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Matlab Software</td>
			<td style="width:71px;">The MathWorks, Inc.</td>
			<td style="width:119px;">Matlab R2012a</td>
			<td style="width:167px;">Code requires the Image Processing Toolbox</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:216px;">Shielded Cable</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SHC68-68</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic technique to probe cell deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

To investigate the deformability of different cell types, human myeloid leukemia cells (HL-60), differentiated neutrophil cells, mouse lymphocyte cells, and human ovarian cancer cell lines (OVCAR8, HEYA8) are evaluated using the &#8216;Cell Deformer&#8217; microfluidic technique. Representative results for the transit time of HL-60 and neutrophil-type HL-60 cells show the timescale for a single cell to transit through a series of constrictions, as shown in Figure 6. Transit time is measured for a populat...

Watch this Scientific Journal Video about A Microfluidic Technique to Probe Cell Deformability at JoVE.com

A Microfluidic Technique to Probe Cell Deformability

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

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