The authors wish to acknowledge support from the Hainan Provincial Natural Science Foundation of China (NO. 320QN211) and the Research Fund Program of Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture of China (NO. PBEA2021ZD01).

All animal protocols during this study were approved by the Hainan University Institutional Animal Care and Use Committee (Protocol number: HNUAUCC-2022-00063; Approval date: 2022-03-03). A list of the equipment and supplies used in this experiment can be found in the Table of Materials. A summary of the current protocol is illustrated in Figure 1.
1. Fish preparation
<ol>
	<li>Obtain 6 month old Nile tilapia with a mean body weight of 100 g from a reliable source. Select fish that are free of any signs of disease.</li>
	<li>Stop feeding the fish 24 h before proceeding to the next step. 
	NOTE: Fish fasting reduces the volume of intestinal contents and facilitates the preparation of intestinal segments.</li>
	<li>Rinse the fish in well-aerated sterile water for 15 min to wash off loosely bound bacteria on the body surface.</li>
	<li>Euthanize the fish with 300 mg/L tricaine methanesulfonate (MS-222) for about 10 min until all movement stops.</li>
</ol>
2. Reagent preparation
<ol>
	<li>Prepare 0.08% BSA-Dulbecco&#39;s phosphate-buffered saline (BSA-DPBS, for cell rinsing and resuspension). Dissolve 0.08 g of BSA (0.8% w/v) in 100 mL of 1x DPBS. Store at 4 &#176;C, and pre-cool on ice when used. 
	NOTE: The addition of BSA is beneficial for minimizing cell clumping. The BSA ratio can be increased from 0.04% to 1% according to the cell clumping conditions.</li>
	<li>Prepare the enzymatic cell dissociation reagent.
	<ol>
		<li>Dilute the collagenase/dispase with 1x PBS to a final concentration of 1 mg/mL (0.1 U/mL collagenase and 0.8 U/mL dispase). Ensure that PBS and not DPBS is used. 
		NOTE: PBS provides the calcium ions needed for the enzyme to function properly. DPBS should not be used in this step because it is Ca2+/Mg2+ free. Additionally, collagenase type I is used in the current protocol.</li>
		<li>Filter the dilution through a sterile 0.22 &#181;m membrane filter.</li>
		<li>Prepare a cell dissociation reagent that is composed of a 95% volume of collagenase/dispase solution and a 5% volume of fetal bovine serum (FBS). 
		NOTE: The dissociation reagent should be made fresh every time it is used. Using 5% FBS helps maintain the cell viability during digestion.</li>
	</ol>
	</li>
	<li>Prepare trypan blue solution.
	<ol>
		<li>Take the trypan blue solution from the 4 &#176;C refrigerator, and keep it at 15-20 &#176;C for a few minutes before use to prevent precipitation.</li>
		<li>Filter the solution through a sterile 0.22 &#181;m membrane.</li>
	</ol>
	</li>
</ol>
3. Equipment preparation
<ol>
	<li>Pre-cool the refrigerated centrifuge to 4 &#176;C before use.</li>
	<li>Obtain sterile RNase-free 1.5 mL centrifuge tubes, 50 mL conical tubes, wide-bore glass pipettes and tips, membrane filters, cell strainers, and all the low retention tubes and tips.</li>
	<li>Autoclave glass pipettes and dissection tools, including forceps, scissors, and scalpels, and pre-treat them with the RNase removal solution to prevent the adverse effects of RNase for single-cell RNA-seq.</li>
</ol>
4. Tissue dissection and cell dissociation
<ol>
	<li>Put the euthanized fish on ice for dissection. Aseptically, collect 3-4 cm of the mid-section of the intestine, and excise as much fat and mesentery as possible. Carefully remove the fat attached to the intestinal surface and the layer of elastic and malleable clear mucosa with forceps. 
	NOTE: The fat and mesentery are attached to the external surface of the intestine.</li>
	<li>Remove as much fat as possible to prevent its adverse effects on the dissociation protocol. Using a syringe, gently rinse the fragments with sterile ice-cold PBS several times to wash off the intestinal contents and mucus. Avoid rough handling.</li>
	<li>Dissect the segment of intestine into small pieces, and transfer them to a 1.5 mL tube filled with 1 mL of ice-cold PBS.</li>
	<li>Centrifuge at 300 &#215; g for 5 min at 4 &#176;C. Remove the supernatant gently, and replace with 1 mL of ice-cold 0.08% BSA-DPBS. Resuspend the tissue fragments by gentle aspiration using a glass pipette. Repeat this step twice.</li>
	<li>Remove the supernatant, and digest the tissue fragments using 1 mL of the enzymatic dissociation reagent (950 &#181;L of collagenase/dispase solution and 50 &#181;L of FBS) in a 37 &#176;C water bath for 30-60 min.
	<ol>
		<li>Additionally, add 5 &#181;L of RNase inhibitor (40 U/&#181;L in the stock solution) to the dissociation reagent if the samples are prepared for single-cell RNA sequencing. 
		NOTE: The final concentration of the collagenase/dispase mix is 1 mg/mL, including collagenase at 0.1 U/mL and dispase at 0.8 U/mL.</li>
	</ol>
	</li>
	<li>Manually invert the tube at 5 min intervals during the 30-60 min water bath. 
	NOTE: The exact incubation time varies according to the tissues used. Check the progress of dissociation under a microscope until no bulk tissue is seen. Place 10 &#181;L of the cell suspension on a glass slide with a pipette, and examine it under the microscope. Increase the digestion time if the cells are not completely dissociated.</li>
	<li>Spin down the tubes at 300 &#215; g for 5 min at 4 &#176;C, and gently remove the enzymatic cell dissociation reagent using wide-bore tips. Add 1 mL of ice-cold 0.08% BSA-DPBS, and gently pipette the cells up and down using a wide-bore glass pipette for 1 min.
	<ol>
		<li>Repeat this step twice. 
		NOTE: Pipetting up and down should be performed with care using wide-bore and low-retention tips to prevent cell disruption.</li>
	</ol>
	</li>
	<li>Pre-wet a 40 &#181;m cell strainer with 0.08% BSA-DPBS, and place it into a 50 mL conical tube. Pass the cell suspension through the strainer. Tap and rinse with an additional 200 &#181;L DPBS, and collect the suspension at the bottom of the tube.</li>
	<li>Transfer the suspension into a 1.5 mL tube. Add 5 &#181;L of DNase I (1 U/&#181;L), and incubate for 15 min at 15-20 &#176;C. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant, and resuspend the cell pellet in 400 &#181;L of ice-cold DPBS (Ca2+/Mg2+ free). Gently pipette up and down with wide-bore tips to mix the cells. Repeat this step twice.</li>
</ol>
5. Staining and microscopic examination
<ol>
	<li>For staining, mix the cell suspension with 0.4% trypan blue solution at a 1:1 ratio. Incubate for 3 min at room temperature.</li>
	<li>Add 5-10 &#181;L of the mixture onto a hemocytometer, and examine under the microscope. 
	NOTE: Viable cells will have unstained cytoplasm, while dead cells will have a blue cytoplasm (Figure 2).</li>
	<li>Count the viable and dead cells in three different fields under the microscope. Calculate the cell viability percentage by dividing the number of viable cells by the total number of cells in a field. 
	NOTE: The cell viability should be more than 85%, and cell concentration should not be less than 1 x 105-1 x 107/mL. The cell suspension background should be clean, without clumps, fragments, or impurities.</li>
</ol>

The authors have no conflicts of interest to disclose.

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine. Before dissociation, the removal of fat and mesentery from the intestine is necessary, particularly for carnivorous fish intestines with much fat. Using a syringe instead of hard scraping to wash off the intestinal contents reduces the mechanical damage to the cells. To ensure cell viability, it is also essential to maintain the temperature at 20 &#176;C or below for the tissue dissection and rinsing steps. The was...

Cells are the fundamental units of organisms. Compared to bulk-tissue studies, single-cell level studies can reflect cell heterogeneity and provide higher-resolution information1. In recent years, researchers have applied single-cell sequencing technologies for genome, transcriptome, epigenome, or multi-omic studies at the single-cell level in mammals, zebrafish, and other model organisms and reported great breakthroughs2,3,4,5,6,7. While most studies have focused on model organisms, there are few reference protocols or commercial dissociation kits for single-cell sequencing in economic fish species, which limits the application of single-cell sequencing in aquaculture research. Therefore, developing tissue dissociation protocols that produce high-quality single-cell suspensions with high cell viability and nucleic acid integrity is crucial.
Optimizing the tissue dissociation protocol with the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. The most effective enzyme for tissue dissociation varies depending on the tissue type. In mammals, several enzymes have been used to prepare single-cell suspensions for mammalian solid tissues, including collagenase, dispase, trypsin, papain, elastase, hyaluronidase, liberase, accutase, and trypLE8,9. Trypsin digestion combined with mechanical disruption has commonly been used to dissociate tissues for cell culture in fish10,11,12,13,14. Trypsin has also been used or added into the digestion cocktail for tissue dissociation in the rat intestine15 and zebrafish gill tissue16. However, for several reasons, trypsin is not the best option for single-cell sequencing. Trypsin alone is usually ineffective for tissue dissociation. Additionally, trypsin induces DNA strand breaks17,18 and RNA degradation19.
Papain degrades the proteins that make up the tight junctions between cells. In mammalian nerve and smooth muscle cells, papain is more efficient and less destructive than other proteases20,21. However, like trypsin, papain results in the free DNA-induced aggregation of cells due to the cell lysis that occurs during enzymatic digestion9. Elastase breaks down elastin, which is typically found in the skin, lungs, ligaments, tendons, and vascular tissues22. It is often used in combination with collagenase, dispase, or trypsin for dissociating lung tissue8. Hyaluronidase cleaves the glycosidic bonds of hyaluronan, contributing to the digestion of the extracellular matrix in various connective tissues and skin9,23.
In general, collagenase and dispase are good options for extracellular matrix breakdown. They have been used in the dissociation of human, mouse, and zebrafish intestines24,25,26,27. Collagenase destroys the peptide bond in collagen, promotes the digestion of the extracellular matrix, and releases the cells into suspension, and, thus, collagenase is often used for human and mouse solid tissue dissociation, including for the liver28,29, spleen30, pancreas31, and intestine25. Dispase is a protease that hydrolyzes the N-terminal peptide bonds of nonpolar amino acid residues and is milder than collagenase. It cleaves the extracellular matrix components, such as fibronectin, type IV collagen, and to a lesser extent, type I collagen, without affecting cell-cell junctions. Dispase is used separately or with other enzymes for tissue dissociation, such as for intestine25,32, brain33, liver34, etc. In addition, commercially available digestion cocktails, including liberase, accutase, and trypLE, are also good alternatives for solid tissue dissociation, especially for the skin, liver, and kidney8,9.
Nile tilapia (Oreochromis niloticus) belongs to the family Cichlidae of the order Perciformes. It is one of the most cultured freshwater fish species in tropical and subtropical areas, with an annual production of 4.5 million tons in 202235. It is one of the best-studied aquaculture fish species with a well-annotated genome. Nile tilapia is an ideal research model for aquaculture fish species due to its short generation time, ease of rearing, and adaptability to a wide range of rearing environments. The intestine is of great research interest as it is the organ of nutrition digestion and absorption, metabolism, and mucosal immunity. The intestine is the habitat of microbial populations and is an essential immune tissue36. It is immunologically active due to the presence of numerous immune cell types, including macrophages, B cells, granulocytes, and T cells.
In the current study, we developed a protocol for preparing a high-quality single-cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. According to the characteristics of these tissue-specific enzymes and preliminary work, collagenase/dispase is appropriate for dissociating tilapia intestine tissue. The final enzyme type to consider in preparing single-cell suspensions is DNase-I, which prevents cell aggregation by degrading free DNA released through dead cell lysis during enzymatic digestion without initiating apoptotic pathways9 and increases the live cell yield36. Additionally, bovine serum albumin (BSA) is added to the washing buffer to reduce cell clumping and improve cell viability. Several reagent companies describe BSA as an enzyme stabilizer. The addition of 0.04%-1% BSA to PBS (phosphate-buffered saline) has been used to develop a washing solution for preparing single-cell sequencing suspensions without adverse effects38. The addition of a low ratio of BSA could help maintain cell viability and avoid the free DNA-induced aggregation of cells due to cell lysis. This protocol can also provide a valuable reference for developing cell dissociation protocols from the intestines of other aquaculture fish species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22-&#956;m Sterile Filter</td>
			<td>Solarbio Life Sciences</td>
			<td>SLGV033RB</td>
			<td>It is used to filter and sterilize the enzyme solution.</td>
		</tr>
		<tr>
			<td>40-&#956;m Cell Strainer</td>
			<td>Solarbio Life Sciences</td>
			<td>F8200</td>
			<td>Cell Strainer is applied to eliminate undigested tissue pieces.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>SRE0098</td>
			<td>Powder; dilute 0.04 g BSA with 100 mL 1&#215; DPBS to prepare 0.04% BSA-DPBS washing bffer. Store at 2 - 8 &#176;C.</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Sangon Biotech</td>
			<td>A004202</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Collagenase/dispase</td>
			<td>Roche</td>
			<td>10269638-001</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Sigma-Aldrich</td>
			<td>D4818</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>AMPD1</td>
			<td>DNase I helps reduce cell clumping.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS), Ca2+/Mg2+-free</td>
			<td>Solarbio Life Sciences</td>
			<td>E607009-0500</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>Sangon Biotech</td>
			<td>A600438</td>
			<td>Dilute with PBS to a final concentration of 0.5 mg/mL.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>Serum, used at volume of 5% in digetstion solution.</td>
		</tr>
		<tr>
			<td>Inverted&#160;Microscope</td>
			<td>Leica</td>
			<td>qTOWER3G</td>
			<td>It is used to examine cell viability.</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td>Dilute with PBS to a final concentration of 0.25 mg/mL.</td>
		</tr>
		<tr>
			<td>Nile tilpia (Oreochromis niloticus)</td>
			<td>ProGift Aquaculture Technology Co. Ltd.</td>
			<td>NA</td>
			<td>Healthy fish with no disease signs (Mean body weight: 100 g).&#160;</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Solarbio Life Sciences</td>
			<td>P1020</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>It is used to spin down the tissue and cell petet.</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>NEB</td>
			<td>M0314L</td>
			<td>Inhibit RNase activity</td>
		</tr>
		<tr>
			<td>Solid-phase RNase-Be-Gone Reagent</td>
			<td>Sangon Biotech</td>
			<td>B644201-0050</td>
			<td>It is used to remove the RNase from tools such as dissecting scissors and glass pipettes. Store at room temperature.</td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>For fish euthanasia.&#160;</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Invitrogen</td>
			<td>C0040</td>
			<td>It is used for staining dead cells.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sangon Biotech</td>
			<td>E607001</td>
			<td>Dilute with PBS to a final concentration of 1 mg/mL.</td>
		</tr>
	</tbody>
</table>

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.

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine for single-cell sequencing (Figure 1). This research shows that the collagenase/dispase mix has a good dissociation effect and is mild for intestine tissue. The selection of the optimal digestion enzyme is essential for preparing a high-quality single-cell suspension. In the preliminary work, the dissociation efficiencies of several commonly used enzymes were compared, and the results a...

Watch this Scientific Journal Video about Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing at JoVE.com

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

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 ...

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1.6K Views.  Hainan University. Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Video: Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

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 ...

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.

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 ...

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 ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...