The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

1. Preparation of Cells
<ol>
	<li>Maintain a culture of a thymic epithelial cell line (IT76M1) or cell to be tested in an incubator (37&#176;C/5% CO2).</li>
	<li>Wash the cells with PBS 1x (repeat this item 3x).</li>
	<li>Add Trypsin to the cells for 5 min.</li>
	<li>Add medium (twice of the volume of trypsin added in item 1.3) with 10% FBS (fetal bovine serum) to the cells with trypsin and centrifuge (800 x g for 5 min).</li>
	<li>Count the cells in a hemocytometer.</li>
	<li>Adjust the density of cells according to the cell type as the cells have to be in close contact with each other to allow coupling. Note: In our case, we used 3 x 105 cells per 35 mm Petri dish.</li>
</ol>
2. Micropipette Preparation
<ol>
	<li>Pull the micropipette as specified from a glass capillary micropipette (1.5 mm diameter) to a final 0.2 &#181;m of diameter so as to attain a final resistance of approximately 30 M&#937;17,18. 
	NOTE: Alternatively, injection pipettes can be purchased. The resistance depends on the cell size, for instance a higher resistance microelectrode would be necessary for pancreatic acinar cells, for example (100-150 M&#937;)19. A common problem that could occur is the precipitation of Lucifer Yellow solution which can then obstruct the micropipette and may require prior filtration or centrifugation. Before injection, the micropipette should be analyzed under the microscope to detect if there is an obstruction or any type of disruption13. The micropipette can be tested by injecting LY with the micropipette tip inside a saline solution.</li>
</ol>
3. Testing the Micropipette
<ol>
	<li>Prepare the Lucifer Yellow solution (5%) in 150 mmol/L LiCI and load the micropipette using a syringe or by backfilling (put into it LY Solution).</li>
	<li>Place the micropipette over the 35 mm Petri dish with the IT76M1 cells on the microinjection workstation and submerge the tip of the glass micropipette into the cell medium. Focus on the micropipette and perform a dye flowing test by applying a pulse.</li>
</ol>
4. Single-cell Lucifer Yellow Microinjection
<ol>
	<li>Focus the microscope right above the cell layer using a high magni&#64257;cation (40X), then slowly lower the pipette to the cells using the micromanipulator.</li>
	<li>Puncture the target cell when the tip is close enough to touch the cell membrane, and apply a small hyperpolarizing pulse to introduce the LY into the cell. The applied voltage will depend on the net charge of dye to be injected.&#160; Alternatively, some other dyes could be used with this technique as shown in Table 1. 
	Note: In principle any hydrophilic dye with MW less than 1KDa could be used. However, the rate of transfer could vary according to the weight and hydrophilicity. Additionally, unspecific transfer of the dye used must be evaluated.&#160;&#160;</li>
	<li>Capture cell images 3 min after dye injection or make a small movie with time lapse microscopy (30 fps). 
	NOTE: A similar approach could be seen in Hitomi et al (2015)20. To avoid communication by intercellular bridges (incomplete mitosis), a co-injection of rhodamin dextran (from 2 to 10 KDa), which does not pass through gap junctions but passes through intercellular bridges and certain types of nanotubules is recommended as shown in Figure 2.</li>
</ol>

<ol>
	<li>Bennett, M. V., 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=Gap+junctions:+new+tools,+new+answers,+new+questions.">Gap junctions: new tools, new answers, new questions.</a> Neuron. 6 (3), 305-320 (1991).</li><li>Orellana, J. A., Martinez, A. D., Retamal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+channels+and+hemichannels+in+the+CNS:+Regulation+by+signaling+molecules.">Gap junction channels and hemichannels in the CNS: Regulation by signaling molecules.</a> Neuropharmacology. , (2013).</li><li>Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+correlates+of+gap+junction+permeation.">Structural correlates of gap junction permeation.</a> Int Rev Cytol. 66, 81-146 (1980).</li><li>Loewenstein, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctional+intercellular+communication+and+the+control+of+growth.">Junctional intercellular communication and the control of growth.</a> Biochim Biophys Acta. 560 (1), 1-65 (1979).</li><li>Alves, L. 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=Functional+gap+junctions+in+thymic+epithelial+cells+are+formed+by+connexin+43.">Functional gap junctions in thymic epithelial cells are formed by connexin 43.</a> Eur.J Immunol. 25 (2), 431-437 (1995).</li><li>Alves, L. 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=Are+there+functional+gap+junctions+or+junctional+hemichannels+in+macrophages?.">Are there functional gap junctions or junctional hemichannels in macrophages?.</a> Blood. 88 (1), 328-334 (1996).</li><li>Fonseca, P. C., 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=Characterization+of+connexin+30.3+and+43+in+thymocytes.">Characterization of connexin 30.3 and 43 in thymocytes.</a> Immunology letters. 94 (1-2), 65-75 (2004).</li><li>Nihei, O. K., 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=Modulatory+effects+of+cAMP+and+PKC+activation+on+gap+junctional+intercellular+communication+among+thymic+epithelial+cells.">Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells.</a> BMC Cell Biol. 11, 3 (2010).</li><li>Czyz, J., Szpak, K., Madeja, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+connexins+in+prostate+cancer+promotion+and+progression.">The role of connexins in prostate cancer promotion and progression.</a> Nat Rev Urol. 9 (5), 274-282 (2012).</li><li>El-Saghir, J. A., El-Habre, E. T., El-Sabban, M. E., Talhouk, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexins:+a+junctional+crossroad+to+breast+cancer.">Connexins: a junctional crossroad to breast cancer.</a> Int J Dev Biol. 55 (7-9), 773-780 (2011).</li><li>Cea, L. 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=Connexin-+and+pannexin-based+channels+in+normal+skeletal+muscles+and+their+possible+role+in+muscle+atrophy.">Connexin- and pannexin-based channels in normal skeletal muscles and their possible role in muscle atrophy.</a> J Membr Biol. 245 (8), 423-436 (2012).</li><li>Cotrina, M. L., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+connexins+in+demyelinating+diseases:+therapeutic+potential+of+glial+targets.">Brain connexins in demyelinating diseases: therapeutic potential of glial targets.</a> Brain Res. 1487, 61-68 (2012).</li><li>Park, H. a. n. -. A., R, S., Khanna, S. a. v. i. t. a., Sen, C. h. a. n. d. a. n. . K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Technologies+in+Single-Cell+Microinjection+and+Application+to+Study+Signal+Transduction.">Current Technologies in Single-Cell Microinjection and Application to Study Signal Transduction.</a> Methods in Redox Signaling. , (2010).</li><li>Abbaci, M., Barberi-Heyob, M., Blondel, W., Guillemin, F., Didelon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+limitations+of+commonly+used+methods+to+assay+the+molecular+permeability+of+gap+junctional+intercellular+communication.">Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication.</a> Biotechniques. 45 (1), 33-52 (2008).</li><li>Stewart, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connections+between+cells+as+revealed+by+dye-coupling+with+a+highly+fluorescent+naphthalimide+tracer.">Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer.</a> Cell. 14 (3), 741-759 (1978).</li><li>Meda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+function+of+connexin+channels+in+primary+tissues.">Probing the function of connexin channels in primary tissues.</a> Methods. 20 (2), 232-244 (2000).</li><li>Klaunig, J. E., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+gap+junctional+intercellular+communication.">Assessment of gap junctional intercellular communication.</a> Curr Protoc Toxicol. , (2009).</li><li>Hanani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lucifer+yellow+-+an+angel+rather+than+the+devil.">Lucifer yellow - an angel rather than the devil.</a> J Cell Mol Med. 16 (1), 22-31 (2012).</li><li>Orci, L., Biochemistry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockage+of+Cell-to-Cell+Communication+within+Pancreatic+Acini+Is+Associated+with+Increased+Basal+Release+of+Amylase+Materials+and+Methods+Preparation+of+Acini.">Blockage of Cell-to-Cell Communication within Pancreatic Acini Is Associated with Increased Basal Release of Amylase Materials and Methods Preparation of Acini.</a> Cell. 103 (August), 475-483 (1986).</li><li>Hitomi, 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=Differential+connexin+function+enhances+self-renewal+in+glioblastoma.">Differential connexin function enhances self-renewal in glioblastoma.</a> Cell Rep. 11 (7), 1031-1042 (2015).</li><li>Nihei, O. K., Campos de Carvalho, ., C, A., Spray, D. C., Savino, W., Alves, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+form+of+cellular+communication+among+thymic+epithelial+cells:+intercellular+calcium+wave+propagation.">A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation.</a> Am J Physiol Cell Physiol. 285 (5), C1304-C1313 (2003).</li><li>Kanno, Y., Loewenstein, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+Diffusion.">Intercellular Diffusion.</a> Science. 143 (3609), 959-960 (1964).</li><li>Alves, L. A., Nihei, O. K., Fonseca, P. C., Carvalho, A. C., Savino, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+modulation+by+extracellular+signaling+molecules:+the+thymus+model.">Gap junction modulation by extracellular signaling molecules: the thymus model.</a> Braz J Med Biol Res. 33 (4), 457-465 (2000).</li></ol>

The authors have no conflicts of interest.

In order to verify the presence of functional intercellular gap junction, the use of tracers, which are membrane impermeable, although permeable by intercellular channels are required16. Fluorescein, the first fluorescent dye to observe cell-to-cell coupling22, is permeable between non junctional membranes3 and has therefore been substituted by Lucifer Yellow dye15. Currently, to find the best choice among the many different t...

Gap junctions are intercellular channels that allow the intercommunication among neighboring cells1. This communication connects two or more neighboring cells, where each one contributes with a connexon or hemichannel to form the intercellular channel. In mammalian cells, the connexon is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm2. Gap junctions not only permit the flow of ions, second messengers and small metabolites, but also contribute to many forms of cellular communication in many physiological processes, such as synaptic transmission, heart contraction, cell growth and differentiation3,4,5,6,7,8. In addition gap junctions have been associated with many diseases including cancer9,10, muscular atrophy11, some genetic diseases and demyelinating diseases12.
This type of intercellular crosstalk can be evaluated by several methods13,14,15,16. In this paper, we show how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. We discuss how to prepare the cells and the micropipette, the usage of the micromanipulator and the injection of Lucifer Yellow dye in a thymic epithelial cell line. Usually, this experimental procedure could be analyzed by the average of connected cells to the cell loaded with dye. In addition, this method could be used with other fluorescent dyes with molecular weight below the gap junctions cut-off which is approximately 1,000 daltons.

<table border="0" cellpadding="0" cellspacing="0" width="1228">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lucifer yellow</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L0259</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lithium Chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L4408</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS tablets</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:137px;">P4417</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">R4130</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Bovine fetal serum</td>
			<td style="width:71px;">Cultilab</td>
			<td style="width:137px;"></td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Trypsin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">T4799</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:137px;">TE-2000</td>
			<td>For microinjection experiments, one needs an inverted fluorescence microscope and &#64257;lters for &#64258;uorescent microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibration-insulated table&#160;</td>
			<td style="width:71px;">Newport</td>
			<td style="width:137px;">VH3036W-OPT</td>
			<td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:137px;">MMO-203</td>
			<td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Current Generator&#160;</td>
			<td style="width:71px;">Digitimer</td>
			<td style="width:137px;">DS2</td>
			<td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&#160;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

single-cell microinjection for cell communication analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Thymic epithelial cell line IT-76MI were used to evaluate dye coupling by gap junctions as these cells were described to express functional gap junctions formed by connexin 4321. Figure 1 shows the injection of Lucifer Yellow when applied in the one cell below the tip of the pipette. After few minutes, connected cells become fluorescent (asterisks) indicating the diffusion of the fluorescent dye through the gap junctions. The number of cells and time to became fluo...

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

single-cell-microinjection-for-cell-communication-analysis

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11.2K Views.  Oswaldo Cruz Foundation. We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Video: Single-cell Microinjection for Cell Communication Analysis

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down to the level of a single cell. Experiments using this method have achieved quantifying over 3,000 proteins across 1,500 single mammalian cells (500-1,000 proteins per cell) in 10 days of mass spectrometer instrument time. SCoPE2 leverages a freeze-heat cycle for cell lysis, obviating the need for clean-up of single cells and consequently reducing sample losses, while expediting sample preparation and simplifying its automation. Additionally, the method uses an isobaric carrier, which aids protein identification and reduces sample losses.
This video protocol provides detailed guidance to enable the adoption of automated single-cell protein analysis using only equipment and reagents that are widely accessible. We demonstrate critical steps in the procedure of preparing single cells for proteomic analysis, from harvesting up to injection to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, viewers are guided through the principles of experimental design with the isobaric carrier, quality control for both isobaric carrier and single-cell preparations, and representative results with a discussion of limitations of the approach.

In this protocol, we describe how to prepare mammalian cells for single-cell proteomics analysis via mass spectrometry using commercially available reagents and equipment, with options for both manual and automatic pipetting.

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the ...