Name: standardized preparation of single-cell suspensions from mouse lung tissue using the gentlemacs dissociator
Uploaded: 2009-07-02T00:00:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator at JoVE.com

The authors are employees of Miltenyi Biotec GmbH, Germany.

To work under sterile conditions, it is recommended to perform all steps in a laminar flow hood.
1. Materials
<ol>
<li>HEPES buffer: 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) </li>
<li>Collagenase D solution: Collagenase D: 100 mg/mL (Collagenase D &gt; 0.15 U/mg), in HEPES buffer </li>
<li>DNase I solution: 20,000 U/mL DNase I </li>
<li>PBS buffer at pH 7.2 </li>
<li>PEB buffer: 1 part BSA Stock Solution in 20 parts autoMACS Rinsing Solution </li>
<li>Tissue: lung derived from 6/7 week old adult female BALB/C mouse, free of adjoining organs.</li>
<li>Optional: CD11c MicroBeads, MACS MS Columns, MACS Separator for the isolation of dendritic cells from mouse lung single-cell suspension</li>
</ol>
2. Dissociating the lung tissue
<ol>
<li>Rinse tissue in a Petri dish containing PBS to remove erythrocytes. </li>
<li>Transfer a maximum of 450 mg lung tissue to a gentleMACS C Tube containing 4.9 mL HEPES buffer.</li>
<li>Add 100 &mu;L Collagenase D solution to a final concentration of 2 mg/ml Collagenase D.</li>
<li>Add 10 &mu;L DNase I solution for up to 150 mg tissue (final concentration of 40 U/ml DNase I) or 20 &mu;L DNase I for 150-450 mg lung tissue (final concentration of 80 U/mL DNase I).</li>
<li>Tightly close the C Tube and attach it onto the gentleMACS Dissociator. Then run the program "m_lung_01". </li>
<li>Incubate for 30 min at 37&deg;C with automated rotation or manually turning every 5 min.</li>
<li>Return sample to the gentleMACS Dissociator and run the program "m_lung_02".</li>
</ol>
3. Filtration
<ol>
<li>Prepare a 50 mL tube for collecting filtered cells by replacing the cap with a 70 &mu;m mesh cell strainer. </li>
<li>Once the second gentleMACS Program ends, depending on the distribution of sample material in the tube, you may centrifuge the tube briefly to collect the sample material at the bottom of the tube. </li>
<li>Remove the cells through the septum sealed cap of the C Tube using a suitable 1000 &mu;l pipette tip and apply them to the cell strainer. </li>
<li>Wash the cell strainer with 5 ml HEPES buffer at RT. </li>
<li>Centrifuge the cells to a pellet in a 50 mL tube at 300xg for 10 min. </li>
<li>Aspirate the supernatant and resuspend the cell pellet in your desired volume of PEB buffer.</li>
</ol>
Part 4: Representative Results:
<img src="/files/ftp_upload/1266/1266fig1.jpg" alt="Figure 1" /> Figure 1. Lung dissociation with the gentleMACS&trade; Dissociator resulted in 92% viable cells. Dead cells were fluorescently stained with propidium iodide (PI) ( right dot plot; left dot plot: no PI staining).
<img src="/files/ftp_upload/1266/1266fig2.jpg" alt="Figure 2" /> Figure 2. Dissociation of mouse lung tissue using the gentleMACS Dissociator results in a high percentage of viable leukocytes and endothelial cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry.
<img src="/files/ftp_upload/1266/1266figure2c.png" alt="Figure 2C" /> Figure 3. Single-cell suspension derived from mouse lung tissue was stained with CD11c-FITC and Anti-mPDCA-1-APC to detect mouse CD11clow m-PDCA-1+ plasmacytoid DCs as well as CD11chigh cells.
 <img src="/files/ftp_upload/1266/1266fig4.jpg" alt="Figure 4" /> Figure 4. Enrichment of dendritic cells using CD11c MicroBeads, a MiniMACS Separator and two MS Columns.

<ol>
	<li>Swanson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3-Ligand,+IL-4,+GM-CSF,+and+Adherence-Mediated+Isolation+of+Murine+Lung+Dendritic+Cells:+Assessment+of+Isolation+Technique+on+Phenotype+and+Function.">Flt3-Ligand, IL-4, GM-CSF, and Adherence-Mediated Isolation of Murine Lung Dendritic Cells: Assessment of Isolation Technique on Phenotype and Function.</a> J. Immunol. 173, 4875-4881 (2004).</li><li>Vermaelen, K., Pauwels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+simple+discrimination+of+mouse+pulmonary+dendritic+cell+and+macrophage+populations+by+flow+cytometry:+Methodology+and+new+insights.">Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: Methodology and new insights.</a> Cytometry Part A. 61A, 170-177 (2004).</li></ol>

The authors are employees of Miltenyi Biotec GmbH who make the instrument used in this article.

In this video, we introduce a new method for the dissociation of mouse lung tissue. We show that a combination of mechanical and enzymatic treatment of lung tissue yielded a high percentage of viable leukocytes and endothelial cells. Specifically, the mechanical disintegration of the tissue was achieved by the gentleMACS Dissociator. The gentleMACS C Tubes include a rotor - stator system, which dissociates tissue in a gentle way. The procedure is controlled by the program settings of the instrument. The settings were optimized to achieve high yield and viability of cells. The derived single-cell suspensions were stained with CD45-PE and CD146-FITC or CD31-FITC and analyzed by flow cytometry to detect leukocytes and endothelial cells. Dendritic cells in the single-cell suspension were stained with CD11c-FITC and Anti-mPDCA-1-APC. Furthermore, dendritic cells from a mouse lung single-cell suspension were enriched using MACS Technology: dendritic cells were magnetically labeled using CD11c MicroBeads and separated using a MiniMACS Separator and MS Columns. 1,2 In general, the combination of our new dissociation protocol with magnetic cell sorting represents a standardized method to obtain specific cell types from lung tissue.

<table><tbody><tr><td>C Tubes</td><td>Miltenyi Biotec</td><td>Order no. 130-093-237</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_765_gentleMACS_C_Tubes.aspx&lt;/a&gt;</td></tr><tr><td>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>Order no. 130-093-235</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>MACSmix&amp;#8482; Tube Rotator</td><td>Miltenyi Biotec</td><td>Order no. 130-090-753</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&lt;/a&gt;</td></tr><tr><td>autoMACS&amp;#8482; Rinsing Solution</td><td>Miltenyi Biotec</td><td>Order no. 130-091-222</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_134_155_autoMACS_Rinsing_Solution.aspx&lt;/a&gt;</td></tr><tr><td>MACS&amp;#174; BSA Stock Solution</td><td>Miltenyi Biotec</td><td>Order no. 130-091-376</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_33_295_MACS_BSA_Stock_Solution.aspx&lt;/a&gt;</td></tr><tr><td>CD11c MicroBeads</td><td>Miltenyi Biotec</td><td>Order No. 130-052-001</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_93_132_CD11c_MicroBeads.aspx&lt;/a&gt;</td></tr><tr><td>CD11c-FITC, CD11c-PE</td><td>Miltenyi Biotec</td><td>Order no. 130-092-026130-091-830</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_329_CD11c.aspx&lt;/a&gt;</td></tr><tr><td>Anti-mPDCA-1-APC</td><td>Miltenyi Biotec</td><td>Order no. 130-091-963</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_325_Anti_mPDCA_1.aspx&lt;/a&gt;</td></tr><tr><td>CD146-FITC</td><td>Miltenyi Biotec</td><td>Order no. 130-092-026</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_417_CD146_LSEC_.aspx&lt;/a&gt;</td></tr><tr><td>CD45-PE</td><td>Miltenyi Biotec</td><td>Order no. 130-091-610</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_331_CD45.aspx&lt;/a&gt;</td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>Order no. 130-093-233</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&lt;/a&gt;</td></tr><tr><td>Art 1000 Reach Pipet Tips</td><td>Molecular BioProducts</td><td>Order no. 2079</td><td>&lt;a href=&quot;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&quot;&gt;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.mbpinc.com/html/products/art/display.html&quot;&gt;http://www.mbpinc.com/html/products/art/display.html&lt;/a&gt;</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator at JoVE.com

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

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 ...

standardized-preparation-single-cell-suspensions-from-mouse-lung

Nanyang Technological University

Research

JoVE Journal

Biology

34.5K Views.  Miltenyi Biotec,GmbH.  中文 日本語 

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

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Immunology and Infection

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 ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

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Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

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Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

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Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Medicine

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Source: Yu, X., et al. <a href="https://app.jove.com/v/60023">Rapid Isolation of Dorsal Root Ganglion Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate macrophages from mouse dorsal root ganglion (DRG) using a mechanical dissociation technique. Upon homogenization of the freshly collected DRG tissue, antibodies against the cell-surface receptors on the macrophages are used to label the cells fluorescently, followed by performing flow cytometry to confirm the presence of macrophages in the sample.

Source: Yu, X., et al. Rapid Isolation of Dorsal Root Ganglion Macrophages. J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate ...

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

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Chapters in this video

Isolation of Mouse Lung Dendritic Cells

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

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation & Characterization of Hoechst^low CD45^negative Mouse Lung Mesenchymal Stem Cells

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

Isolation of CD146⁺ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mouse Wound Models and Preparation of Single-Cell Suspensions

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation