We thank members of the Dr. Sabine Costagliola and Singh lab for comments on the manuscript. This work was supported by the Fonds de la Recherche Scientifique-FNRS under Grant number 34772792 &#8211; MISU to S.P.S.

All the procedures presented below were performed in accordance with institutional (Universite&#769; Libre de Bruxelles (ULB)) and national ethical and animal welfare guidelines and regulation, which were approved by the ethical committee for animal welfare (CEBEA) from the Universite&#769; Libre de Bruxelles (protocols 578N-579N).
1. Preparation before tissue dissection
<ol>
	<li>Prepare 0.2% Tricaine solution in PBS for euthanizing the zebrafish. Chill the solution on ice.</li>
	<li>Prepare a 30 mm Petri dish for mincing the tissue.</li>
	<li>Prepare ice cold 1x PBS (10 mL per tissue sample).</li>
	<li>Cool the centrifuge to 4 &#176;C.</li>
	<li>For nuclei isolation, use a detergent-free nuclei isolation kit (Table of Materials).</li>
	<li>Before starting the protocol, pre-chill buffer A and B provided in the kit by placing them on ice for at least 30 min prior to nuclei isolation.</li>
	<li>For handling the isolated nuclei, coat the plastic reagents such as tubes and pipette tips with 5% BSA solution. For this, prepare the solution by dissolving 2 g of BSA in 40 mL of PBS. Coating plastic items with 5% BSA reduces nuclei sticking to plastic. This step enhances the recovery of isolated nuclei.</li>
	<li>Coat the pipette tips by pipetting 5% BSA solution 2-3 times. Air-dry the tips for 2 hours. Prepare 10 plastics tips per sample.</li>
	<li>Coat the tubes for collection of nuclei by filling them with 5% BSA. Invert the tubes 3 times to ensure an efficient coating. Remove the solution and air-dry the tubes upside down on a clean tissue paper for 2 hours. Per sample, coat one collection tube provided in the kit. Additionally, prepare coated 1.5 mL tubes for nuclei collection after FACS. 
	NOTE: Glass tips are highly recommended alternative to the plastic pipette tips to minimize the sticking.</li>
</ol>
2. Dissection of zebrafish brain
<ol>
	<li>For euthanizing the zebrafish, prepare a 90 mm Petri dish with 25 mL of ice-cold Tricaine solution.</li>
	<li>Carefully, take the zebrafish from the tank using a fishing net and place it into the Petri dish.</li>
	<li>Euthanize the fish by leaving it in Tricaine for 5 min.</li>
	<li>Decapitate the animal with a sharp razor blade.</li>
	<li>Using forceps, gently break open the skull and remove soft tissues, skin and bones from ventral and dorsal side of the skull.</li>
	<li>Gently, transfer the brain into a fresh 30 mm dish containing ice cold PBS.</li>
	<li>Mince the brain into small pieces using a razor blade to ease the loading of the sample on the spin column.</li>
</ol>
3. Single nuclei isolation
<ol>
	<li>Transfer the minced tissue to the filter cartridge provided in the nuclei isolation kit and add 200 &#181;L of cold buffer A to sensitize the tissue. Grind the tissue using the plastic rod provided by the kit for 2 min.</li>
	<li>Add 300 &#181;L of cold buffer A and incubate the filter cartridge on ice with cap open for 10 min.</li>
	<li>Cap the cartridge and resuspend the tissue by inverting the tube 5 times.</li>
	<li>Centrifuge at 16,000 x g for 30 s. In this step, cells are ruptured when passing through the filter and high-speed centrifugal force is applied. The flow through contains intact nuclei, which pellet at the bottom of the tube.</li>
	<li>Discard the filter and resuspend the pellet by vortexing vigorously for 10 s.</li>
	<li>Pellet the nuclei by centrifuging the solution at 500 x g for 3 min. Discard the supernatant carefully as the nuclei pellet is colorless.</li>
	<li>Resuspend the pellet in 0.8 mL of cold buffer B and centrifuge at 600 x g for 10 min. In this step, nuclei are separated from membrane debris. The colorless pellet obtained contains isolated nuclei.</li>
	<li>Resuspend the isolated nuclei in 500 &#181;L of PBS with 5% BSA. Keep the nuclei suspension on ice to perform FACS after the quantification.</li>
</ol>
4. Visualization of nuclei morphology
<ol>
	<li>Confirm nuclear morphology by Hoechst staining. For this, remove 100 &#181;L of single nuclei suspension in a new tube using BSA coated tips. To stain the nuclei, add 0.1 &#181;L of Hoechst (1 mg/mL). Gently vortex the tube.</li>
	<li>Transfer the nuclei suspension to glass bottom dish for imaging.</li>
	<li>Image the nuclei using a fluorescence microscope with laser excitation settings of ~405 nm (Violet) wavelength.</li>
</ol>
5. FACS based enrichment of nuclei
<ol>
	<li>Before performing FACS, filter the nuclei using a 40 &#181;m cell strainer into BSA coated tube.</li>
	<li>Dilute the filtered suspension by adding PBS with 5% BSA to a final volume of 1,000 &#181;L.</li>
	<li>Label two round bottom FACS tube as &#8216;control&#8217; and &#8216;stained&#8217;. The &#8216;control&#8217; tube will contain un-stained nuclei, while the &#8216;stained&#8217; tube will have Hoechst stained nuclei.</li>
	<li>Transfer 250 &#181;L of the nuclei suspension into &#8216;control&#8217; tube using BSA coated pipette tip.</li>
	<li>Transfer the remaining 750 &#181;L solution to FACS tube labeled as &#8216;stained&#8217; and add 1 &#181;L of Hoechst dye to stain the nuclei. Mix by slow vortexing.</li>
	<li>Load the unstained control sample to cell sorter. Record 5000 events.</li>
	<li>Load the stained samples and record 5000 events.</li>
	<li>Draw FACS gates that allows identification of single nuclei. Nuclei can be selected by comparing the Hoechst fluorescence signal between control and stained sample.</li>
	<li>Sort Hoechst-positive nuclei from the stained tube into new 1.5 mL tube containing 50 &#181;L of PBS with 5% BSA. 
	NOTE: Isolated nuclei can be collected into a desired medium according to requirements of the downstream application.</li>
</ol>

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The authors have no conflict of interest. The payment for making the article open access was made by Invent Biotechnologies Inc., USA.

Profiling the transcriptome and epigenome at a single-cell resolution has revolutionized the study of biological systems. Studies at the resolution of a single cell for a solid tissue depend on dissociation of the organ into individual cells or nuclei. Dissociation is a destructive procedure that can introduce technical artifacts, which can prevent development of an accurate representation of the system5,6. For instance, enzymatic dissociation can harm cells with complex morphologies, such as neurons or podocytes, and can induce expression of stress and heat-shock response genes7,12. Additionally, use of detergent during dissociation can rupture the nuclear membrane and lead to aggregation23,25. Thus, optimizing the dissociation to obtain a single cell or nuclei suspension of the highest quality is paramount to the success of high-throughput profiling experiments.
Here, we demonstrate a detergent and enzyme-free nuclei isolation method that allows extraction of intact nuclei from zebrafish brain in less than 20 minutes. The protocol yields nuclei with typical morphology and robust integrity (Figure 2). From a single zebrafish brain weighing 6 mg, the protocol yields a total of 60,000 nuclei determined by a hemocytometer count. The isolated nuclei can be utilized for multiple downstream applications, including snRNA-seq, ATAC-seq and immunostaining. The isolated nuclei may include cross-contamination from cytoplasmic fractions, particularly from components of endoplasmic reticulum and mitochondria. For high-throughput profiling experiments, clearance of cellular debris, particularly mitochondria, is strongly recommended. Flow cytometry (Figure 3) provides a viable option for purification of nuclei. Alternatively, the sucrose gradient can also be utilized for removal of debris.
The protocol has been tested on mouse thyroid gland (data not shown) and provides results similar to zebrafish brain tissue. Overall, the protocol provides a robust, reproducible, and universal method for preparation of single nucleus suspension, helping to simplify logistics for high-throughput profiling experiments.

Single-cell RNA-seq (scRNA-Seq) and ATAC-seq are versatile tools to study complex biological systems at single-cell resolution. They are widely utilized to define cell subtypes and states, gene networks and to assess cellular heterogeneity. A prerequisite for performing scRNA-seq is the preparation of a single cell suspension by tissue dissociation. Due to the variation in the extracellular matrix composition and mechanical properties, individual tissues require optimization of the dissociation protocol for preparation of single cell suspension.
Dissociation of tissues into single cells typically involves treatment with digestive enzymes, including collagenase, dispase or trypsin, at 37 &#176;C1,2,3,4. As transcriptional machinery remains active at 37 &#176;C, enzymatic dissociation can introduce mRNA expression artifacts and noise5,6. Notably, prolonged incubation can induce stress responsive genes and heat-shock response in a non-uniform manner &#8211; leading to technical variability in the experiment7.
Another drawback of generating a single cell suspension is the difficulty in obtaining viable and intact cell-types with complex morphologies. In particular, neurons, adipocytes and podocytes are challenging to isolate8,9,10,11. For instance, Wu and colleagues demonstrated the absence of glomerular podocytes in scRNA profiles from an adult mouse kidney12. Similar nonoptimal observations have been made regarding the recovery of interconnected neurons from brain tissue8,13,14. In sum, dissociation protocols can introduce detection bias towards easier to dissociate cell-types, leading to a misrepresentation of the cellular architecture of the organ.
To overcome the technical noise and bias introduced during sample preparation in scRNA-Seq., isolation and profiling the nucleus provides an attractive alternative. As nuclear morphology is similar between different cell-types, isolation of the nuclei circumvents the issue of isolating intact and viable cells with complex morphologies. For instance, Wu and colleagues demonstrated successful profiling of glomerular podocytes with the single-nucleus RNA-Seq. (snRNA-Seq.) of an adult mouse kidney, which was missing from scRNA-Seq12. Intriguingly, comparative studies between single-cell and single-nucleus RNA-seq have suggested a decrease in induction of stress and heat-shock response genes with snRNA-Seq12. The studies further suggest a high correlation between the genes detected by the two methods. However, a recent study on human microglia failed to detect genetic activation in Alzheimer&#8217;s disease15. Thus in certain contexts, snRNA-Seq is a suitable alternative for scRNA-Seq16,17. Additionally, the nuclear isolation can be utilized for single-cell ATAC-Seq., providing information about the regions of open-chromatin within individual cells.
The protocol for nuclei isolation involves three major steps: i) detergent-based lysis of cell membrane to release the nucleus; ii) tissue homogenization using a Dounce homogenizer; and iii) enrichment of nuclei and removal of cell debris using gradient centrifugation or flow cytometry18,19,20,21,22. Among this, the first two steps depend on the tissue type and need to be empirically optimized. Mild detergent leads to partial rupture of cell membrane and inefficient retrieval of nuclei from the tissue23. On the other hand, high level of detergent and harsh homogenization leads to rupture of the nuclear membrane and their loss24,25. Ruptured nuclei further tend to clump together and form aggregates, which if not removed can lead to artifacts in the downstream profiling experiment.
To circumvent the issues related to detergent optimization for nuclei isolation, we introduce a protocol to isolate intact nuclei from fresh samples using a detergent-free and spin-column-based method. The protocol yields nuclei from whole organ within 20 minutes, limiting the induction of artifactual transcription. The isolated nuclei can be enriched with FACS for single-nuclei RNA-Seq. and ATAC-seq, providing a simple and universal method that enables robust and reproducible high-throughput profiling.

<table>
	<tbody>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Carl Roth</td>
			<td>90604-29-8</td>
			<td>Albumin fraction V</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>FACSAria III</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sartorius</td>
			<td>A-14C</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes (1.5 mL)</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon (15 mL)</td>
			<td>Corning</td>
			<td>352096</td>
			<td>Polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>Falcon (5 mL )</td>
			<td>Corning</td>
			<td>352052</td>
			<td>Polystyrene round bottom test tubes</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi cell strainer (40 &#956;m)</td>
			<td>Sigma</td>
			<td>BAH136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle (250 mL)</td>
			<td>VWR</td>
			<td>215-1593</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottomed dish</td>
			<td>World Precision Instruments</td>
			<td>FD3510-100</td>
			<td>Fluorodish 35 mm</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>VWR</td>
			<td>612-1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipette socket</td>
			<td>Carl Roth</td>
			<td>388.1</td>
			<td>Pipetting aid pi-pump 2500</td>
		</tr>
		<tr>
			<td>Hoechst staining dye solution</td>
			<td>Abcam</td>
			<td>ab228551</td>
			<td>Hoechst 33342</td>
		</tr>
		<tr>
			<td>Minute Detergent-Free Nuclei Isolation Kit</td>
			<td>Invent Biotechnologies</td>
			<td>NI-024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>ThermoFisher</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (30 mm)</td>
			<td>FisherScientific</td>
			<td>11333704</td>
			<td>Pyrex</td>
		</tr>
		<tr>
			<td>Petri dish (90 mm)</td>
			<td>Corning</td>
			<td>758-10178-CS</td>
			<td>Gosselin</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>VWR</td>
			<td>89079</td>
			<td>10 &#956;L, 200 &#956;L, 1000 &#956;L</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Gilson</td>
			<td>F167380</td>
			<td>Pipetman</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Swann-Morton</td>
			<td>7981809</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methane sulfonate</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex machine</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Vortex-Genie 2</td>
		</tr>
	</tbody>
</table>

nuclei isolation from whole tissue using a detergent and enzyme-free method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

--The protocol described above was used to generate single nucleus suspension directly from zebrafish brain tissue. The isolation typically requires 20 minutes and avoid the use of detergent or digestive enzyme. A schematic summarizing the individual steps of the protocol is provided in Figure 1, which can be printed to be used for guidance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61471/61471fig01.jpg" /> 
Figure 1: Schematic of detergent-free spin-column-based method for nuclei isolation. 
Graphical representation of the individual steps performed during extraction of nuclei from fresh zebrafish brain tissue. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Visualization of nuclear morphology
For qualitative confirmation of the nuclear morphology, the isolated nuclei were stained with Hoechst and visualized using fluorescence microscopy. The nuclei appeared intact, round and well-separated (Figure 2). Importantly, nuclear aggregation, a sign of nuclear membrane rupture, was absent.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61471/61471fig02.jpg" /> 
Figure 2: Single nuclei isolation from zebrafish brain. 
Fluorescence microscopy image of Hoechst-stained nuclei demonstrating their intact morphology. Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
FACS-based enrichment of the intact nuclei
Enrichment of isolated nuclei and removal of cellular debris was performed by flow cytometry by gating on the presence of a Hoechst fluorescence signal. The Hoechst signal was detected upon excitation with violet, 405 nm, laser (Brilliant Violet 421 &#8211; BV421). Unstained nuclei displayed background fluorescence (Figure 3A, Supplementary Figure 1A), while stained nuclei exhibited strong fluorescent signal (Figure 3B, Supplementary Figure 1B). As illustrated in Figure 3C, the unstained and Hoechst stained nuclei were well segregated in the violet channel.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61471/61471fig03.jpg" /> 
Figure 3: Isolated nuclei display strong and specific Hoechst fluorescent signal in flow cytometry. 
Histogram plots for single nuclei suspension displaying the distribution of Hoechst staining. Hoechst is excited by violet, 405 nm, laser (Brilliant Violet 421 &#8211; BV421). The unstained sample (A) displays signal in the range of 100-103, while Hoechst stained nuclei (B) emit signal in the 103-105 range. An overlay of fluorescence intensity emitted by unstained (grey) and stained (blue) samples (C) demonstrates clear separation between the two populations. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61471/61471supfig01v2.jpg" /> 
Supplementary Figure 1: Flow cytometry gating strategy for isolated nuclei. Representative flow plots for isolated nuclei suspension. Isolated nuclei were analyzed using forward scatter and Violet&#160;laser BV421 which excites Hoechst at 405 nm. The unstained sample (A) displayed BV421 signal in the 100-103 range. Out of 13130 events, 141 events were detected as single nuclei based on FSC-A (1.07% of total), and 0 events for unstained nuclei based on BV421 signal (0% of total). The Hoechst stained nuclei (B) displayed BV421 signal in the 103-105 range. Out of 50000 events, 2418 events were detected as single nuclei based on FSC-A (4.84% of total), and 2414 events for Hoechst-positive nuclei based on BV421 signal (4.83% of total). <a href="https://cloudflare.jove.com/files/ftp_upload/61471/61471supfig01.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method at JoVE.com

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

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24.2K Views.  Université Libre de Bruxelles (ULB). Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Video: Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...