Introduction to Equipment
<ol>
<li>Tissue is gently dissociated, using violet C Tubes into viable single-cell suspensions.</li>
<li>The PBS/BSA EDTA buffer is used to dissociate the cells. The buffer is prepared by adding 1 part of BSA Stock solution to 20 parts of autoMACS Rinsing Solution containing EDTA. </li>
<li>The MACS Pre-Separation Filters are used so that the dissociated tissue samples are free of all cell clumps, promoting successful MACS separation and subsequent analysis. </li>
</ol>
Dissociating the spleen cells
<ol>
<li>Whole spleens are disscociated into single-cell suspensions of splenocytes using the gentleMACS Dissociator.</li>
<li>To begin the procedure, load the C Tube with a volume of buffer according to the mass of the spleens. A healthy spleen of a young adult female BALB/c mouse weighs about 80-120 mg. Every 1 to 2 spleens of this mass require 3 mL of buffer. Spleens from older animals or animals with infections are often larger and must be weighed in buffer. The buffer volumes must be scaled accordingly. Do not exceed a total buffer volume of 10 mL.</li>
<li>Add the dissected spleen to the C Tube containing 3 ml of buffer.</li>
<li>Make sure that the C Tube cap is tight and attach the C Tube to the dissociator with the cap down.</li>
<li>Power up the dissociator. For 1-2 spleens, select program m_spleen_01. The program will take 56 seconds to complete. If the number of spleens is greater, use program m_spleen_04; this program can be used for up to 720 mg of spleen tissue.</li>
<li>After completion of the program the native spleen tissue is dissociated into a splenocyte suspension.</li>
<li>Spin down the whole cell suspension at 300xg at room temperature for about 30 sec. This will ensure that rotor and stator are freed of all cells which are collected at the bottom of the C Tube.</li>
<li>Remove the sample through the septum-sealed opening in the C Tube cap using a 1000 microliter pipetter. </li>
</ol>
Filtration step
<ol>
<li>To begin the filtration step, apply the dissociated tissue to the Pre-Separation Filter or a cell strainer with 30 &micro;m pore size. The strainer should fit a 15 mL tube for 1-2 spleens or a 50 mL tube for more than 2 spleens.</li>
<li>Let the cell suspension pass through. Then wash the filter by applying 5 mL of buffer.</li>
<li>Now, centrifuge the tube with the cell suspension at 300xg, at room temperature for 10 minutes to collect all splenocytes in a spleen cell pellet.</li>
<li>Now, that we have a pellet of dissociated splenocytes, we must aspirate the supernatant and resuspend the cell pellet in an appropriate amount of buffer.</li>
</ol>
Spleen dissociation using the gentleMACS Dissociator will yield a high percentage of viable splenocytes, which is demonstrated by flow cytometry using the MACSQuant Analyzer. Dead cells are stained by propidium iodide. In the example provided, the forward side scatter dot plot shows the prepared splenocytes.

All protocols and data sheets are available at <a href="http://www.miltenyibiotec.com">http://www.miltenyibiotec.com.</a>.

To avoid tedious and often painful manual dissociations, the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions. The tissue dissociation procedure is gentle to cells and yields cell suspensions with a high viability rate. The gentleMACS Dissociator in combination with C Tubes is designed for the preparation of single-cell suspensions from various tissues. The instrument is equipped with a number of pre-set programs to dissociate mouse spleen, liver, lung, or neural tissue. The range of programs and protocols will be continuously expanded to include additional tissues.

<table><tbody><tr><td>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>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>C Tubes</td><td>Miltenyi Biotec</td><td>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>13 mL Tubes</td><td>Miltenyi Biotec</td><td>130-091-596</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_751_13_mL_Tubes_for_LS_Columns.aspx&lt;/a&gt;</td></tr><tr><td>autoMACS&amp;#8482; Rinsing Solution </td><td>Miltenyi Biotec</td><td>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>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>Pre-Separation Filters </td><td>Miltenyi Biotec</td><td>130-041-407</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&lt;/a&gt;</td></tr><tr><td>MACSQuant Analyzer</td><td>Miltenyi Biotec</td><td>130-092-197</td><td></td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>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></tbody></table>

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.

Watch this Scientific Journal Video about Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

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37.7K Views.  Miltenyi Biotec,GmbH.  中文 JapaneseVersion 

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

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Medicine

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.

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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 ...

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Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

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Source: Nguyen, H. D., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp. (2020).
This video describes T-cells enrichment from the spleen of a mouse by negative selection using a magnetic separator. The technique involves targeting the other cells in the sample for depletion using antibodies or ligands directed against specific cell surface antigens.

Source: Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cancer Research

Leukemia

Cell isolation & characterization

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Source: Yu, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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 ...

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

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