Name: counting proteins in single cells with addressable droplet microarrays
Uploaded: 2018-07-06T13:00:00
Description: Watch this Scientific Journal Video about Counting Proteins in Single Cells with Addressable Droplet Microarrays at JoVE.com

ASR designed experiments, developed protocols and analyzed data. SC and PS performed cell size experiments. ASR and OC wrote the manuscript. The authors wish to gratefully acknowledge the support of Prof. David R. Klug for providing access to equipment. The authors wish to thank the Imperial College Advanced Hackspace for access to fabrication and prototyping facilities.

1. Preparation
<ol>
	<li>Make chips and print antibody microarrays
	<ol>
		<li>Attach an adhesive silicone/acrylic isolator to a coverslip functionalized to support an antibody microarray. This is referred to as the chip. 
		NOTE: Various surface chemistries have been tested for their suitability with addressable droplets.5 Surface chemistries may need to be optimized for alternative capture agents. ADM isolators are available commercially or may be produced by laser cuttin...

<ol>
	<li>Willison, K. R., Klug, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+single+cell+and+single+molecule+proteomics+for+clinical+studies.">Quantitative single cell and single molecule proteomics for clinical studies.</a> Curr. Opin. Biotechnol. 24 (4), 745-751 (2013).</li><li>Heath, J. R., Ribas, A., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+tools+for+drug+discovery+and+development.">Single-cell analysis tools for drug discovery and development.</a> Nat. Rev. Drug Discov. 15 (3), 204-216 (2016).</li><li>Eyer, K., Stratz, S., Kuhn, P., K&#252;ster, S. K., Dittrich, P. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressable+droplet+microarrays+for+single+cell+protein+analysis.">Addressable droplet microarrays for single cell protein analysis.</a> Analyst. 139 (21), 5367-5374 (2014).</li><li>Cristofanilli, M., Budd, G., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> N. Engl. J. Med. 351 (8), 781-792 (2004).</li><li>Lan, F., Haliburton, J. R., Yuan, A., Abate, A. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+drug+resistance+properties+in+scarce+cancer+stem+cells+using+droplet+microarray.">Elucidating drug resistance properties in scarce cancer stem cells using droplet microarray.</a> Nano Today. 7 (1), 29-34 (2012).</li><li>Popova, A. A., Demir, K., Hartanto, T. G., Schmitt, E., Levkin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-microarray+on+superhydrophobic-superhydrophilic+patterns+for+high-throughput+live+cell+screenings.">Droplet-microarray on superhydrophobic-superhydrophilic patterns for high-throughput live cell screenings.</a> RSC Adv. 6 (44), 38263-38276 (2016).</li><li>Chen, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+nanoinjection+for+high-thoughput+chemiluminescence+immunoassay+on+multicapillary+glass+plate.">Inkjet nanoinjection for high-thoughput chemiluminescence immunoassay on multicapillary glass plate.</a> Anal. Chem. 85 (15), 7413-7418 (2013).</li><li>Zhu, Y., Zhu, L. -. N., Guo, R., Cui, H. -. J., Ye, S., Fang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter-scale+protein+crystallization+and+screening+with+a+microfluidic+droplet+robot.">Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.</a> Sci. Rep. 4, 5046 (2014).</li><li>Sun, Y., Chen, X., Zhou, X., Zhu, J., Yu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-in-oil+array+for+picoliter-scale+analysis+based+on+sequential+inkjet+printing.">Droplet-in-oil array for picoliter-scale analysis based on sequential inkjet printing.</a> Lab Chip. 15 (11), 2429-2436 (2015).</li><li>Liberski, A. R., Delaney, J. T., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="One+cell-one+well":+A+new+approach+to+inkjet+printing+single+cell+microarrays.">"One cell-one well": A new approach to inkjet printing single cell microarrays.</a> ACS Comb. Sci. 13 (2), 190-195 (2011).</li><li>Yusof, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet-like+printing+of+single-cells.">Inkjet-like printing of single-cells.</a> Lab a Chip - Miniaturisation Chem. Biol. 11 (14), 2447-2454 (2011).</li><li>Zhu, Y., Zhang, Y. -. X., Liu, W. -. W., Ma, Y., Fang, Q., Yao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+2-dimentional+droplet+array+for+single-cell+reverse+transcription+quantitative+PCR+assay+with+a+microfluidic+robot.">Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.</a> Sci. Rep. 5, 9551 (2015).</li><li>Ueda, E., Geyer, F. L., Nedashkivska, V., Levkin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+Microarray:+facile+formation+of+arrays+of+microdroplets+and+hydrogel+micropads+for+cell+screening+applications.">Droplet Microarray: facile formation of arrays of microdroplets and hydrogel micropads for cell screening applications.</a> Lab Chip. 12 (24), 5218-5224 (2012).</li><li>Kozak, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-volume+wall-less+immunoassays+using+patterned+planar+plates.">Micro-volume wall-less immunoassays using patterned planar plates.</a> Lab Chip. 13 (7), 1342-1350 (2013).</li><li>Yen, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembled+Pico-Liter+Droplet+Microarray+for+Ultrasensitive+Nucleic+Acid+Quantification.">Self-Assembled Pico-Liter Droplet Microarray for Ultrasensitive Nucleic Acid Quantification.</a> ACS Nano. 9 (11), 10655-10663 (2015).</li><li>Au, A. K., Lai, H., Utela, B. 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Interface. 5, S113-S121 (2008).</li><li>Salehi-Reyhani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+advantages+and+constraints+in+miniaturized+capture+assays+for+single+cell+protein+analysis.">Scaling advantages and constraints in miniaturized capture assays for single cell protein analysis.</a> Lab Chip. 13 (11), 2066-2074 (2013).</li><li>Brown, R. B., Audet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+techniques+for+single-cell+lysis.">Current techniques for single-cell lysis.</a> J. R. Soc. Interface. 5, S131-S138 (2008).</li><li>Womack, M. D., Kendall, D. A., MacDonald, R. 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Addressable Droplet Microarrays are a sensitive and extensible method for quantitatively determining the absolute copy number of protein within a single cell.
Limiting the level of non-specific binding (NSB) is critical within the protocol to achieving as low a limit of detection as possible. Proteins and other biochemical species may non-specifically bind to a number of interfaces present within the droplets &#8212; the coverslip surface, the antibody spot and the oil/water interface. Protein...

The goal of this method is to determine the variation in abundance of a target protein in a cell population with single cell resolution. Single cell analysis provides a number of benefits that are not available with traditional ensemble biochemical methods.1,2,3,4,5 Firstly, working at the single cell level can capture the rich heterogeneity of a cell population that would otherwise be lost by the averaging that occurs with traditional ensemble biochemical techniques. The majority of work-horse biochemical methods work with the bulk, requiring, as they often do, millions of cells to produce a result. Of course, the consequences of assessing entire cell populations depends on a number of factors, for example, the heterogeneity in protein expression where some important features of the distribution of protein abundance may be missed. From a practical perspective, the sensitivity required of single cell techniques make them capable of working with amounts of biological material that is insufficient for even the more sensitive bulk techniques to function. A key example of this is the study of rare cell types such as circulating tumor cells (CTCs) where even for patients with a poor prognostic outlook less than 10 CTCs might be present in a single 7.5 mL blood draw.6 Here we present the methodology required to perform single cell protein measurements using a reduced volume antibody-based assay employing oil-capped droplets printed on an antibody microarray.
Microfluidic droplet platforms are high throughput, able to generate thousands of droplets per second, and capable of isolating, and even culturing, single cells in individual droplets to perform a wide array of biochemical assays. Droplet-based techniques are well suited for single cell analysis,7,8,9 with notable recent examples including DropSeq10 and inDrop11, which have been greatly aided by the power of amplification techniques. The limited amount of material and no methods of amplification for proteins make single cell proteomics especially challenging.
Droplets may be analyzed by a number of methods and fluorescence microscopy has been widely used. Single molecule techniques such as total internal reflection fluorescence (TIRF) microscopy allows fluorescent molecules to be visualized with unparalleled signal-to-noise ratio.12 Due to the exponential decay of the evanescent field, only fluorophores in high proximity to the surface (order of 100nm) are excited making TIRF a good strategy in detecting small amounts of a target molecule in a complex mixture. The inherent optical sectioning strength of TIRF also helps to avoid wash steps and limits assay time and complexity. However, TIRF requires planar surfaces and examples of TIRF microscopy applied to droplets in flow involve the formation of a planar surface of which to image.13 To this end, single cell proteomic techniques often design microfluidic chips around surface-immobilized capture agents in a microarray format.4,14
The droplets, themselves, may be formed in arrays on planar surfaces, so-called droplet microarrays.15,16,17 Spatially organizing droplets into arrays allows them to be conveniently indexed, easily monitored over time, individually addressed and, if required, retrieved. Droplet microarrays can achieve a high density of micro-reactors with thousands of elements per chip which are either free-standing or supported by microwell structures.18,19,20 They may be formed by sequential deposition by liquid handling robots, inkjet spotters, contact microarrayers21,22,23,24,25,26 or they can self-assemble on surfaces such as superhydrophillic spots patterned on a superhydrophobic surface.27,28,29
With these considerations in mind, Addressable Droplet Microarrays (ADMs) were designed to combine the versatility, spatial addressability and reduced volumes of droplet microarrays with the sensitivity of single molecule TIRF microscopy to quantitatively measure protein abundance.5 ADMs enable single cell analysis forming a droplet microarray containing single cells over an antibody microarray, which is then capped with oil to prevent evaporation. The volumes of the droplets are discrete to prevent sample loss, which would otherwise be achieved by on-chip valving in continuous flow microfluidics.30 The absolute amount of target protein from a single cell is extremely small; however, the reduced volume of the droplets allows for relatively high local concentration in order that they are detected using a sandwich antibody assay - antibody is immobilized in a distinct region, or spot, on a surface which captures protein which in turn binds to a fluorescently labelled detection antibody present in the droplet volume. As a label-free approach (i.e. protein targets do not need to be labelled directly), ADMs are generally applicable to analyzing cells from primary sources, such as processed blood, fine need aspirates and dissociated tumor biopsies, as well as cells from culture and their lysates.
Measuring the variation in protein abundance across a cell population is important in determining the heterogeneity in response, for example, to a drug and will help in providing insight into cellular functions and pathways, assessing subpopulations and their behavior as well as identify rare events that would otherwise be masked by bulk methods. This protocol describes how to produce and use addressable droplet microarrays to quantitatively determine the abundance of the transcription factor p53 in human cancer cells and may be used to investigate the role of p53 in response to chemotherapeutic drugs. The target protein is determined by the choice of capture and detection antibodies and may be modified to include more or different targets. Instructions are provided to build a simple apparatus incorporating a concentric nozzle from general lab consumables to manually array 10 nL droplets capped with oil. The full experimental process is described whereby each droplet is then loaded with a single cell, which is then lysed and the expression of protein determined with single molecule resolution using TIRF microscopy.

<table>
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010015</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS)</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture flasks</td>
			<td>Corning</td>
			<td>SIAL0639</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Biochrom</td>
			<td>L2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microarray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontact Arrayer</td>
			<td>DigiLab, UK</td>
			<td>OmniGrid Micro</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontact pin</td>
			<td>ArrayIt, USA</td>
			<td>946MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (Nexterion)</td>
			<td>Schott, Europe</td>
			<td>1098523</td>
			<td>Size (mm): 65.0 x 25.0; Thickness (mm) 0.17</td>
		</tr>
		<tr>
			<td>p53 capture antibody</td>
			<td>Enzo</td>
			<td>ADI-960-070</td>
			<td></td>
		</tr>
		<tr>
			<td>p53 detection antibody, Alexa Fluor 488 labelled</td>
			<td>Santa Cruz</td>
			<td>sc-126</td>
			<td>stock concentration 200&#956;g/mL</td>
		</tr>
		<tr>
			<td>Saline-sodium citrate buffer</td>
			<td>Gibco</td>
			<td>15557-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betaine</td>
			<td>Sigma</td>
			<td>61962</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>384 well plate (low volume)</td>
			<td>Sigma</td>
			<td>CLS4511</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen gas cylinder</td>
			<td>BOC</td>
			<td>Industrial grade, oxygen-free</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Droplets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Eppendorf</td>
			<td>Patchman NP2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Microinjector</td>
			<td>Eppendorf</td>
			<td>CellTram Vario</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Origio, Denmark</td>
			<td>MBB-FP-L-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>KD Scientific</td>
			<td>KDS-210</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L syringe</td>
			<td>Hamilton</td>
			<td>81020</td>
			<td>Gas tight, PTFE Luer lock</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Hamilton</td>
			<td>81327</td>
			<td>Gas tight, PTFE Luer lock</td>
		</tr>
		<tr>
			<td>Silicone isolator</td>
			<td>Grace Bio-Labs</td>
			<td>JTR24R-A-0.5</td>
			<td>6x4 well silicone isolator with adhesive</td>
		</tr>
		<tr>
			<td>Laser cutter</td>
			<td>VersaLASE</td>
			<td>VLS2.30 CO2 Laser 3W</td>
			<td>for laser cutting of custom isolators</td>
		</tr>
		<tr>
			<td>1mm thick acrylic sheet</td>
			<td>Weatherall-UK</td>
			<td>Clarex Precision Sheet 001</td>
			<td>for laser cutting of custom isolators</td>
		</tr>
		<tr>
			<td>Adhesive sheet</td>
			<td>3M</td>
			<td></td>
			<td>used to adhere custom isolators to microarrayed coverslips</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Loctite</td>
			<td>LOCPFG3T</td>
			<td></td>
		</tr>
		<tr>
			<td>150 &#956;m ID/360 &#956;m OD fused silica tubing</td>
			<td>IDEX</td>
			<td>FS-115</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 mm ID/1/16&#8221; OD PFA tubing</td>
			<td>IDEX</td>
			<td>1503</td>
			<td></td>
		</tr>
		<tr>
			<td>0.014&#8221; ID/0.062&#8221; OD PTFE tubing</td>
			<td>Kinesis</td>
			<td>008T16-100</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 mm ID/2.0 mm OD FEP tubing</td>
			<td>IDEX</td>
			<td>1673</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumen (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP9700100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure water</td>
			<td>Millipore, Germany</td>
			<td>MilliQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy &#38; Optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF microscope with encoded XY stage</td>
			<td>Nikon, Japan</td>
			<td>Nikon Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>EM-CCD</td>
			<td>Andor Technologies, Ireland</td>
			<td>IXON DU-897E</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser excitation source</td>
			<td>Vortran, USA</td>
			<td>Stradus 488-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical lysis laser source</td>
			<td>Continuum, USA</td>
			<td>Surelite SLI-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope filter cube for TIRF</td>
			<td>Chroma, USA</td>
			<td>z488bp</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope filter cube for Optical Lysis</td>
			<td>Laser 2000, UK</td>
			<td>LPD01-532R-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>Open Source</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>version 7.14 or higher</td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

counting proteins in single cells with addressable droplet microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

The absolute basal protein copy number of p53 was determined with single cell resolution in a human colon cancer cell line, BE cells. We demonstrate how p53 expression can vary over several orders of magnitude and show a weakly positive correlation between cell size and protein copy number within the resting BE cell population.
Addressable Droplet Microarrays are formed when aqueous droplets are dispensed at antibody spot locati...

Watch this Scientific Journal Video about Counting Proteins in Single Cells with Addressable Droplet Microarrays at JoVE.com

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

The overall goal of this experiment is to determine the absolute protein copy number of a target protein with single-cell resolution. Single cell techniques will enable a better understanding of the behavior of a protein as it relates to a central dogma and help toward accurate quantitation of the controlled gene expression. Working at the single-cell level can capture the rich heterogeneity of the cell population that would otherwise be lost by the averaging that occurs with traditional biochemical techniques.Generally, individuals new to this method will struggle because of the sensitivity of single-cell assays or a limited amount of protein in each cell. We first had the idea for this method when we wished to perform a single-cell protein assay that could be easily sampled for further analysis down the stream. Begin this procedure by making chips and printing antibody microarrays as described in the text protocol.To prepare the apparatus, feed a 100-millimeter length of 150-micron inner diameter 360-micron outer diameter fused silica tubing to a 40-millimeter length of 1-millimeter inner diameter 1/16-inch outer diameter PFA tubing until it protrudes by two millimeters. This will form the concentric nozzle. Apply a thin layer of cyanoacrylate glue to the end of a 10-microliter pipette tip and insert it into the 40-millimeter piece of PFA tubing.If required, reposition the fused silica tubing to maintain a 2-millimeter protrusion at the nozzle before the adhesive sets. Insert the other end of the fused silica capillary into the end of a 200-millimeter length of 014-inch inner diameter 062-inch outer diameter PTFE tubing. Connect this to a 100-microliter Hamilton syringe filled with four percent bovine serum albumen in phosphate-buffered saline.Next insert a 400-millimeter length of 1-millimeter inner diameter 2-millimeter outer diameter FEP tubing into a 200-microliter pipette tip until it forms a seal. Apply a thin layer of cyanoacrylate glue to another 200-microliter pipette tip, then push this into the first tip to fix the tubing in place. Then connect the open end of the FEP tubing to a 1-milliliter Hamilton syringe filled with mineral oil.Insert the 200-microliter pipette tip assembly into the 10-microliter pipette tip of the concentric nozzle. Next, fill the 100-microliter syringe with 4-percent PBSA blocking solution. Reattach and flush the aqueous tubing with the blocking solution.Repeat twice for a total of three flushes. Then fill the 100-microliter syringe with 0.125-micrograms-per-liter detection antibody in 4-percent PBSA and reattach the tubing. Replace the blocking solution in the tubing by dispensing 25 microliters of detection antibody solution.Flush the oil tubing with mineral oil until all tubing and the nozzle fills with oil. Then refill the 1-milliliter syringe with mineral oil and reattach the tubing. Finally, secure the tubing and nozzle assembly to an XYZ manipulator.To form addressable droplets, secure the chip on the microscope stage. Record the microscope's stage coordinates of each spot in the array using the automated microscope control software. Next, set the XYZ manipulator on the microscope stage.Place the syringes in separate syringe pumps as they will need to be operated independently. Place the nozzle at an angle of 50 to 60 degrees. Then set the aqueous syringe pump to dispense 10 nanoliters of 100 microliters per minute, and set the oil syringe pump to disperse 5 microliters at 100 microliters per minute.Dispense the aqueous solution until a bead of fluid is visible at the head of the nozzle. Dab the bead with a dust-free wipe to remove it. Using the automated microscope control software, set the microscope stage coordinates to an antibody spot in the array and focus on the cover slip surface.When aligning the nozzle, be careful to not disturb the antibody spot. Take extra care and make sure you observe the approach of the glass capillary tip through a microscope. Being careful not to disturb the spot, use the XYZ manipulator to align the glass capillary tip of the concentric nozzle to the side of the antibody spot.Once aligned, dispense 10 nanoliters of aqueous solution. Without moving the stages, dispense five microliters of oil to cap the aqueous solution. Slowly raise the nozzle clear of the droplet and move to the next well.Repeat this step for all antibody spots in the array. After 30 minutes, image all spots in the array using the automated microscope control software to determine the background of single molecules bound to each antibody spot prior to loading cells. To load the addressable droplets with cells, we suspended the cells in a solution of 0.125-micrograms-per-milliliter detection antibody in L-15 media with 10 percent FDS.Filter the cell solution through a 40-micron-pitch cell strainer to ensure a single-cell suspension. Load the single cells into the addressable droplets from the cell reservoir using the micromanipulator and microcapillary. Observing through a 10x objective, use the microinjector to aspirate a single cell into the microcapillary from the cell reservoir.Working manually with a joystick, pour automatically using the eject feature of an electronic manipulator stage. Reject the micropipette by translating it upwards to clear the 1-millimeter height of the chip. Set the stage coordinates to that of an addressable droplet using the automated microscope control software.Inject the micropipette by returning it to the stored Z position. Perform this manually with the joystick or automatically if using an electronic manipulator stage. The mechanical processes due to laser-induced lysis can create an emulsion at the droplet edge that higher-pulse energies can completely disrupt the droplets.Ensure proper lysis pulse energies are kept to a minimum and cells are not deposited at the immediate edge of the droplets. Dispense the cells in the addressable droplet using the microinjector. Image the cells in droplets using bright-field microscopy, including any fluorescence imaging.Also image all spots in the array using single-molecule TIRF microscopy. Focus on the cell in an addressable droplet. Achieve complete optical lysis by focusing a single 6-nanosecond laser pulse close to the location of the cell.Note the time at which each cell is lysed. Acquire single molecule images using TIRF microscopy of all spots every ten minutes for the first 30 minutes, then every 20 minutes for a further 60 minutes. If only interested in the amount of protein bound at equilibrium, image all spots after incubating the chip for 90 minutes at room temperature.To perform single-molcule counting for any non-congested antibody spot, load the background image acquired before lysis. In Fiji, select the background image. Under the image menu, select duplicate to duplicate the background.Then select the duplicate image and under the process filters tab select Gaussian blur and specify a 50-pixel radius. Navigate to process and select image calculator. There specify the operation divide, the images required, then select the boxes create new window and 32-bit result.Subtract each pixel in the image by one. Then select a 50-pixel-by-50-pixel area in any of the four corners in the background flat image. Measure the pixel intensity standard deviation by selecting analyze and measure.Under image adjust, select threshold and set the lower threshold level to three standard deviations. In the segmented image SM mask set the pixel intensity values to zero for any objects that do not have a size of four to nine square pixels and a circularity of 0.5 to 1. To do so, select analyze followed by analyze particles and specify the size and circularity.For the same antibody spots, load and repeat the data analysis steps for all image frames captured as a time-result series acquired post-lysis. In representative results of single-molecule images, signal in the background images shows the lower level of nonspecific binding of the fluorescently labeled detection antibody to the surface and antibody spot. The magnified inset image shows highlighted single molecules with red arrows.Also shown is a typical image of a pulldown of p53 in BE human colon carcinoma cells. This sequence of images shows accumulation of p53 protein on the antibody spot. A calibration curve obtained using known concentrations of the combinant protein is used to convert single-molecule counts on each spot to the number of target proteins in the analysis volume and hints at single-cell copy number.The horizontal red-dashed line indicates the level of nonspecific binding. The basal p53 protein expression in BE cancer cells shows a long-tailed gamma-like distribution where p53 protein expression in some cells is significantly higher than the modal value. Here, p53 protein expression is plotted as a function of cell volume as calculated from the measured cell diameter prior to lysis.The results indicate that a minimum concentration of p53 protein is maintained in these cells. After watching this video, you should have a good understanding of how to measure the abundance of a target protein with single-cell resolution. While working with biological cells, it's important to remember that the proteins can degrade.Once mastered, this technique can be completed within four hours if it is performed properly, and can be incorporated into protocols analyzing primary patient samples. Single-cell techniques can help provide insight into the cellular function and behavior as well as identify rare events that would otherwise be missed by both methods. Though this method can provide insight into the biology of p53 and breast cancer cell lines, it can also be applied to other studies of diseases without the need for labeling, which is beneficial in studying primary cells from patient samples.Following this procedure, other methods like quantum dot PCR may be performed to answer additional questions such as determining whether target RNA and protein expression is correlated. Measuring the absolute copy number of a protein will be attractive to biomathematicians and computational scientists in supporting their biological models.

Research

JoVE Journal

Cancer Research

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Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

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In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

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Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

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