Name: Generation of Single-Cell Suspensions from Mouse Neural Tissue (Video) | JoVE Journal
Uploaded: 2009-07-07T19:00:00.000+00:00
Duration: 11 min 17 s
Description: Learn to generate single-cell suspensions from mouse neural tissue using the gentleMACS Dissociator. This protocol preserves cell viability and surface proteins for accurate downstream analysis.

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To work under sterile conditions, it is recommended to perform all steps in a laminar flow hood.
1. Materials
<ul>
<li>Neural Tissue Dissociation Kit (P) (Components: Solution 1,2 , 3, and 4, Storage Buffer)</li>
<li>Pre-Separation Filters</li>
<li>(Optional) Myelin Removal Beads</li>
<li>Beta-mercaptoethanol</li>
<li>gentleMACS Dissociator</li>
<li>C Tubes</li>
<li>MACSmix Tube Rotator</li>
<li>HBSS (w/o)</li>
<li>HBSS (w)</li>
</ul>
<ol>
<li>Prepare the following solutions prior to beginning the protocol:<ol>
<li>Hanks’ Buffered Salt Solution without divalent cations Ca2+ or Mg2+ (HBSS (w/o)</li>
<li>HBSS, standard, i.e. with Ca2+ and Mg2+ (HBSS (w)</li>
</ol></li>
<li>Depending on the antigen epitope of interest in subsequent applications, use either the Papain-based Neural Tissue Dissociation Kit (P) or the Trypsin-based Neural Tissue Dissociation Kit (T).</li>
<li>Prepare the following solutions from the Neural Tissue Dissociation Kit:<ol>
<li>Solution 2: add beta-mercaptoethanol to 0.067 mM</li>
<li>Solution 4: dissolve powder in 0.7 ml Storage Buffer (provided in kit), gently mix (do not vortex)</li>
<li>Enzyme Mix 1: Pipette 1.9 mL Solution 2 and 50 μl Solution 1 (both from kit) into a C Tube and incubate for 10-15 min at 37 °C. This is sufficient to dissociate 400 mg of brain tissue.</li>
</ol></li>
</ol>
2. Dissociating the neural tissue
<ol>
<li>Weigh brain tissue in a tube containing 1 ml HBSS (w/o)</li>
<li>Transfer mouse brain into the C Tube containing 1950 μL of preheated Enzyme Mix 1. (NOTE: this protocol is written for ≤ 400 mg, but solution volumes can be scaled to accommodate up to 1600 mg of brain tissue per C Tube)</li>
<li>Place the C Tube onto the gentleMACS Dissociator and run program “m_brain_01”.</li>
<li>Incubate with rotation (4 rpm using a MACSmix Tube Rotator) for 15 min at 37 °C.</li>
<li>Place the C Tube onto the gentleMACS Dissociator and run program “m_brain_02”.</li>
<li>During the dissociation step prepare 30 μl of Enzyme Mix 2 per 400 mg of tissue by gently mixing 20 μl of Solution 3 and 10 μl of Solution 4.</li>
<li>Add Enzyme Mix 2 to the C Tube via the septum-sealed cap and mix gently without vortexing.</li>
<li>Incubate the C Tube with rotation for 10 min at 37 °C in an incubator.</li>
<li>Place the C Tube onto the gentleMACS Dissociator and run program “m_brain_03”.</li>
<li>Incubate the C Tube with rotation for 10 min at 37 °C in an incubator.</li>
<li>Centrifuge briefly to collect the sample at the bottom of the tube.</li>
</ol>
3. Filtration
<ol>
<li>Select a filter large enough to allow passage to the cells of interest. For instance, Purkinje cells are too large to pass through a 30 μm filter, but Prominin-1+ cells will.</li>
<li>Use a suitable1000 μl pipette to remove the cells form the C Tube through the septum-sealed cap and apply it to the Pre-Separation Filter on a 15 ml collection tube. (NOTE: for larger sample sizes use a 50 ml collection tube).</li>
<li>Wash the filter with 10 ml of HBSS (w).</li>
<li>Centrifuge the filtered cells at 300 x g for 10 min at RT.</li>
<li>Resuspend the supernatent in your medium or buffer of choice for subsequent applications.</li>
<li>To remove myelin debris, use Myelin Removal Beads.</li>
</ol>
4. Representative Result: Please See Figures 1-3
<img src="/files/ftp_upload/1267/Fig_1.png" alt="Flow cytometry dot plot, propidium iodide vs. FL-2, cell viability, 97% live, 3% dead cells." data-altupdatedat="1761735611000" data-alt="fig 1"> Figure. 1 The brain dissociation with the gentleMACS Dissociator resulted in 97% viable cells, as flow cytometric analysis shows.
<img src="/files/ftp_upload/1267/1267fig2.png" alt="Microbial colony growth observed under a microscope, illustrating cell culture proliferation." data-altupdatedat="1761735611000" data-alt="fig 2"> Figure. 2 Light microscope picture of neurosphere formation after magnetic cell sorting using Anti-Prominin-1 MicroBeads after 7 days of cultivation in MACS® NeuroMedium supplemented with MACS Supplement B27 PLUS. Cells were prepared from CD1 mouse brain (P3) using the Neural Tissue Dissociation Kit (P).
<img src="/files/ftp_upload/1267/1267fig4.jpg" alt="Prominin-1+ cell isolation flow cytometry scatter plots; data analysis, myelin removal effect." data-altupdatedat="1761735611000" data-alt="fig 3.5"> Figure. 3 Myelin debris in single-cell suspensions considerably impairs cell isolation, and removal of myelin debris by Myelin Removal Beads increases efficiency cell separations. For MACS Separations using Anti-Prominin-1 MicroBeads, P22 mouse brain was dissociated using the Neural Tissue Dissociation Kit (P). Cells from the single-cell suspension were either directly used for separation, or were submitted to myelin depletion using Myelin Removal Beads. Comparing the separation from samples without and with myelin removal, demonstrates that the purity is higher for samples with previous myelin removal.

<ol><li>Hardt, O., Scholz, C., Küsters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li>
<li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li>
<li>Környei, Z., Gócza, E., Rühl, R., Orsolits, B., Vörös, E., Szabó, B., Vágovits, B., Madarász, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li>
<li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li>
<li>Skog, J., Würdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T. Jr, Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li>
</ol>

The authors are employees of Miltenyi Biotec GmbH, Germany.

The gentleMACS Dissociator facilitates the standardized preparation of single-cell suspensions from neural tissues in a closed system. Neural Tissue Dissociation Kits are optimized to preserve antigen epitopes needed for further applications like immunostainings and immunomagnetic cell separation1-5. In this protocol we show the gentle enzymatic dissociation of mouse brain using the gentleMACS Dissociator and the Neural Tissue Dissociation Kit (P), yielding in 97% viable cells. 100 mg of neural tissue yields between 5x106 and 1x107 cells, depending on the age of the host tissue. The targeted Prominin-1+ cells were isolated using Anti-Prominin-1 MicroBeads and subsequently taken into culture. The protocol is shown to be even more effective with the additional use of Myelin Removal Beads when working with neural tissue derived from mice &gt;P7.

<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_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.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>Neural Tissue Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>Order no. 130-092-628</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&lt;/a&gt;</td></tr><tr><td>Pre-Separation Filters</td><td>Miltenyi Biotec</td><td>Order no. 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>Anti-Prominin-1 MicroBeads</td><td>Miltenyi Biotec</td><td>Order no. 130-092-333</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&lt;/a&gt;</td></tr><tr><td>Anti-Prominin-1-APC</td><td>Miltenyi Biotec</td><td>Order no. 130-092-335</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&lt;/a&gt;</td></tr><tr><td>Myelin Removal Beads</td><td>Miltenyi Biotec</td><td>Order no. 130-094-544</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.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><tr><td>Neural Tissue Dissociation Kit (T)</td><td>Miltenyi Biotec</td><td>Order no. 130-093-231</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.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></tbody></table>

generation of single-cell suspensions from mouse neural tissue

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 ...

Within the nervous system. Hundreds of neuronal 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 gentle max associator when used in combination with trypsin or pepane based dissociation kits can effectively and gently dissociate brain tissue while preserving antigen epitopes in limiting cell loss. Once generated single cell suspensions can be treated with monoclonal conjugates like anti prominent one microbeads, which identify neuro progenitors or purified further using myelin removal beads.Hi, my name is Sandra PeNAT. I'm a project manager for neuroscience products at Mil Biotech here in baggage Gabert Germany. Today I will show you how to prepare a single cell suspension from mouse brain tissue using the genix dis associator.The genix dis associator is a device that can dissociate a variety of tissue types in a closed sterile environment, and so it eliminates the need for tedious manual mechanical dissociation. So let's get started. Depending on the antigen epitope of interest in subsequent applications use either the papain based neural tissue dissociation kit or the trypsin based neural tissue dissociation kit.For today's experiment, we prefer the neuro tissue dissociation kit P as we would like to isolate prominent positive cells using anti prominent one microbeads. Prominent one is neither sensitive to pepane nor to trypsin, and in such a case, the neuro tissue dissociation kit P is the better choice. Before the protocol is initiated, the reagents and solutions must be prepared for this kit.Have on hand your own stock of Hank's balance salt solution HBSS without calcium ions and magnesium ions HBSS with calcium and magnesium ions and beam me capto. Ethanol begin by preparing solution two solution four and subsequently enzyme mix one. First, add betta me capto ethanol to solution two to a final concentration of 0.067 millimolar.The solution is now stable for up to one month when stored at four degrees Celsius. Next, dissolve the lyophilized powder and a vial labeled solution.Four. Using 0.7 milliliter of storage buffer mix gently and do not vortex.Now prepare enzyme mix one by pipetting 1900 microliters of solution two and 50 microliters of solution one in into a C tube. This is enough solution to dissociate 400 milligrams of tissue, so if more will be dissociated, prepare additional enzyme mix one. A maximum of 1600 milligrams of brain tissue can be processed in a C tube.The enzyme mix one in the C tube is then incubated at 37 degrees Celsius for 10 to 15 minutes. To begin the dissociation pipette one milliliter of HBSS without calcium and magnesium ions into the tube. Set the tube on a balance, add the brain tissue and record its mass.This P four mouse brain weighs about 0.2 grams. An adult brain would weigh about 0.4 grams for all tissue samples less than 400 milligrams. The solution volumes we will use today are adequate.If your tissue weighs more than 400 milligrams than scale up all reagent volumes accordingly, up to 1600 milligram tissue can be processed in one C tube. Transfer the mouse brain into the prepared C tube containing 1, 950 microliters of preheated and enzyme. Mix one and seal the cap tightly.Now place the C tube onto the gen max associator with the cap down and make sure that the tissue is located in the area of the stator. Select the program MCO brain O one and run the program. This is the first of three programs you will run on the dissociation when the program finishes.Remove the C tube and incubate the sample for 15 minutes at 37 degrees Celsius under slow continuous rotation. Using the max mix tube rotator, the running mode of the device corresponds to four RPM. After the first incubation, put the tube back onto the dissociation upside down and run program M brain O2.During the next association, prepare 30 microliters of enzyme mix two per 400 milligrams of tissue by adding 20 microliters of solution, three to 10 microliters of solution four.When the second associator cycle has come to an end at 30 microliters of enzyme mix two into the C tube directly through the septum sealed opening in the center of the cap. To ensure sterility, invert the C tube gently to mix and do not vortex. Then put the C tube back on the max mix tube rotator and incubate the sample for 10 minutes at 37 degrees Celsius with four RPM mixing.Then the CTU is returned onto the gentle max dis associator for the third and last time, and the program M brain oh three is run. When the third program is completed, put the C tubes on the rotator and incubate for 10 more minutes at 37 degrees Celsius. Now centrifuge the sample briefly to collect the cells at the bottom of the tube.We are now ready to filter the cells. Choose a cell strainer according to the size of your cell type of interest. If the cells you want to collect are greater than 30 micrometers in diameter such as kinji cells, the pore size must be large enough to pass cell bodies of this diameter.For prominent one positive cells, a 30 micrometer filter will work. Remove the dissociated tissue from the C tube using 1000 microliter pipette tips that fit through the septum sealed opening in the C tube cap. Apply the cell suspension to the pres separation filter placed on a 15 milliliter collection tube.For larger sample sizes of 50 milliliter tube may be required. Allow the cell suspension to run through the filter. Then wash the filter by adding 10 milliliters of HBSS solution with calcium and magnesium ions.When working with tissue quantities greater than 400 milligrams, use up to 30 milliliters of HBSS with calcium and magnesium ions for the washing of the filter. Now pellet the cells in the collection tube by centrifugation at 300 Gs for 10 minutes at room temperature, aspirate the supernat completely and resuspend the cell pellet in your buffer or medium of choice. At this point, you may choose to remove any myelin debris as it can impair cell isolation and antibody staining.To remove myelin debris, we suggest using myelin removal beads. Otherwise, the cells are ready for further application. This dot plot based on propidium iodide staining indicates that 97%of cells present in a single cell suspension from home mouse sprain like the suspension generated in this video are viable.These cells can be further purified using anti prominent microbeads to yield neuronal progenitors, which when cultured in max neuro medium, supplemented with B 27 plus can form neurospheres in these dot plots. The benefit of treating suspensions with the myelin removal beads is observed. A significantly higher percentage of prominent one expressing neuronal progenitors is obtained by max separation following myelin depletion from a single cell suspension.I have just shown you how to prepare single cell suspension from mouse brain tissue using the genix dissociation and the neural tissue dissociation kit. When doing this procedure, it's important to have all solutions prepared appropriately for the right choice of the neural tissue dissociation kit, T or P.Please refer to the NDK application note or data sheet. Using this method, embryonic as well as the D tissue sections or whole brain can be processed under sterile conditions in a time saving and standardized manner.Thank you for watching and good luck for your experiments.

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Learn to generate single-cell suspensions from mouse neural tissue using the gentleMACS Dissociator. This protocol preserves cell viability and surface proteins for accurate downstream analysis.

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