Name: single-cell microinjection for cell communication analysis
Uploaded: 2017-02-26T13:00:00
Description: Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

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 acc...

<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>

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 ...

Hi, my name is Anael. I work at Oswaldo Cruz Foundation at Cellular Communication Laboratory. We study here P2 Receptors and gap injection in the context of immune system and liver.Today, I will present you the microinjection of cell. Gap injections play some physiological roles as synapse transmission, heart contractions, and others. For this technique, we can study if the gap injections are functional or not.Okay, let's see the base components of the microinjection setup. Here we have the inverted fluorescence microscope where we will see the cells. This is a CCD camera to record the results.We have here the current generator that we will introduce the dye into the cell. Next we have the holder where the microelectrode is connected. And the micro-manipulator that's connected to the holder and allow the movement of the microelectrode in the three axis, Z, Y, and X.Before start the experiment, we have to make the microelectrode.We use this borosilicate glass capillary and this equipment named polar. We attach the borosilicate capillary here. This tungsten filament we will heat to make two microelectrodes.Now the tungsten filament's heating and the wave here we will pull down to make the two microelectrodes.Easy. Our first step is to fill the microelectrode with the dye. One tip here is that you have to fill only the tip with a few microliters to do the experiment.It's not necessary to fill everything. Note with more details. Fill only the tip.Let's start an experiment. We have here the cells in the petri dish, in a sodium solution. The silver wire connected to the current generator.We have now to attach the microelectrodes in the head stage. Here in the head stage we have another silver wire also connected to the current generator. Once the microelectrode is attached, we can put it inside of the bath.Note that the tip of the pipette is carefully touching the bath. This procedure is make very carefully to avoid the crash of the tip in the bottom of the petri dish. Once inside of the bath, we can go to the microscope to look for the shadow of the micropipette.We recommend we start looking in the magnification lens. When we find the shadow of the pipette, we can move the microelectrode toward the cell using the micromanipulator. Once it's very close to the cell, we can do a test post to verify if the microelectrode's not obstructed.How we will see next. Here we are seeing the micropipette and we are adjusting the micropipette very close to the cell. We can see cell movement right and left.Then we change to the filter, fluorescence filter. We can apply hyperpolarizing pulse to test if the tip of the pipette is not obstructed. Since the micropipette is inside of the cell, we can apply hyperpolarizing pulse to load the cell with the dye.We use here the loose free yellow dye. After loading the cell with the dye, if the neighbor cells are connected by gap injections, we wait some minutes and we will see neighbor cells brightening by the diffusion of the dye by these gap injections. In our experiment we can see five cells, marked with stars, showing fluorescence.Here there is a good example of an experiment of microinjection in thymic epithelial cells, in A and B.In the figures C and D, shows a microinjection in thymic inert cell of loose free yellow and another dye, not permeable to gap injection showed in the insert. The figure E and F thymic epithelial cell line microinjected with loose free yellow and in the insert loose free yellow and a gap injection block. The results show that loose free yellow did not spread to the neighbor cells.Next we see a microinjection in thymic epithelial cells, in presence of dexamethasone and the quantification in B.Dexamethasone increased the degree of coupling, as showed in the figure B.Of 100 injections, the number of three or four cells connected was more frequent in the presence of this drug. I hope I could help beginners to do this important technique to cellular communication study. Thanks and bye bye.

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

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Biology

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 Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Two-Step Cell Digestion of Mouse Carotid Artery

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Author Spotlight: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

Transcriptome Profiling for High-Throughput Drug Screening

Cost-Efficient Transcriptomic-Based Drug Screening

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

Author Spotlight: Cost-Effective Transcriptomic Drug Screening - Unlocking New Targets 

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

Transcriptomics allows&#160;to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease ...

Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of &#946;-Cell Mitophagy

Begin by culturing the eyelet samples isolated from regular fat diet or high fat diet mice overnight at 37 degrees Celsius. Design the desired experimental conditions for the study. For each experimental condition, using a pipette, pick 100 eyelets into a six centimeter Petri dish containing two milliliters of eyelet medium.To induce Mitophagy, treat the eyelets in the appropriate Petri dishes with two microliters of 250 nano molar Valinomycin stock for three hours. Isolate the eyelets and using a pipette, transfer the eyelets from each Petri dish to a separate micro centrifuge tube. Then, spin the eyelets at 350 G for one minute at 10 degrees Celsius and using a pipette, discard the supernatant.Wash the obtained sample twice with one milliliter of PBS. Between the washes, spin the sample and discard the supernatant as demonstrated previously. To dissociate eyelets into single cells, add 500 microliters of 0.05%Trypsin prewarmed to 37 degrees celsius, to one sample tube.Pipette the contents of the tube up and down repeatedly until the eyelets are visibly dispersed. Immediately add one milliliter of prewarmed eyelet medium to neutralize trypsin. Spin the samples at 500 G for five minutes at 10 degrees Celsius.Remove the supernatant carefully without disturbing the pellet. Then, wash the samples two times with eyelet flow medium. The cell samples are now ready to be stained for flow cytometric analysis of beta cell mitophagy.