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 ...

Research

JoVE Journal

Cancer Research

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

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

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...