Name: combined mechanical and enzymatic dissociation of mouse brain hippocampal tissue
Uploaded: 2021-10-21T13:30:00
Description: Watch this Scientific Journal Video about Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue at JoVE.com

We thank Aimee Rogers for providing hands-on training and continued product support. We thank Dr. Amanda Burke for ongoing troubleshooting and clarifying discussions. We thank Meredith Joheim and the UAMS Science Communication Group for the grammatical editing and formatting of this manuscript.&#160;This study was supported by NIH R25GM083247 and NIH 1R01CA258673 (A.R.A).

Experiments were conducted in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee at UAMS. 6-month-old female C57Bl6/J wild-type mice were purchased and group-housed (4 mice per cage) under a constant 12 h light/dark cycle.
1. Preparation of reagents
<ol>
	<li>Prepare fixable live/dead stain stock solution. Reconstitute the fluorescent stain with 20 &#181;L of dimethyl sulfoxide (DMSO).</li>
	<li>Wrap the vial in foil, label it as &#34;Reconstitu...

<ol>
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L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mammalian+neural+stem+cells+and+neurogenesis:+Five+decades+later.">Adult mammalian neural stem cells and neurogenesis: Five decades later.</a> Cell Stem Cell. 17, 385-395 (2015).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+microglia+on+neurogenesis.">Effects of microglia on neurogenesis.</a> Glia. 63, 1394-1405 (2015).</li><li>Mu, Y., Lee, S. W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+in+adult+neurogenesis.">Signaling in adult neurogenesis.</a> Current Opinion in Neurobiology. 20, 416-423 (2010).</li><li>Fares, J., Diab, Z. B., Nabha, S., Fares, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+hippocampus:+history,+regulation,+and+prospective+roles.">Neurogenesis in the adult hippocampus: history, regulation, and prospective roles.</a> International Journal of Neuroscience. 129, 598-611 (2018).</li><li>Tosun, M., Semerci, F., Maletic-Savatic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stem+cells+in+the+hippocampus.">Heterogeneity of stem cells in the hippocampus.</a> Advances in Experimental Medicine and Biology. 1169, 31-53 (2019).</li><li>Abbott, L. C., Nigussie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+the+mammalian+dentate+gyrus.">Adult neurogenesis in the mammalian dentate gyrus.</a> Journal of Veterinary Medicine Series C: Anatomia Histologia Embryologia. 49, 3-16 (2020).</li><li>Rodrigues, G. M., Fernandes, T. G., Rodrigues, C. A., Cabral, J. M., Diogo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+human+induced+pluripotent+stem+cell-derived+neural+precursors+using+magnetic+activated+cell+sorting.">Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.</a> Methods in Molecular Biology. 1283, 137-145 (2015).</li><li>Ko, I. K., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+analysis+of+multiple+surface+markers+expressed+on+rat+neural+stem+cells+using+antibody+microarrays.">Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays.</a> Biomaterials. 26, 4882-4891 (2005).</li><li>Jager, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+enzymatic+and+mechanical+methods+of+dissociation+on+neural+progenitor+cells+derived+from+induced+pluripotent+stem+cells.">Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells.</a> Advances in Medical Sciences. 61 (1), 78-84 (2017).</li><li>Volovitz, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-aggressive,+highly+efficient,+enzymatic+method+for+dissociation+of+human+brain-tumors+and+brain-tissues+to+viable+single-cells.">A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.</a> BMC Neuroscience. 17, 1-10 (2016).</li><li>Adult brain dissociation kit: mouse and rat. Miltenyi Biotec Available from: <a target="_blank" href="https://www.miltenyibiotec.com/upload/assets/IM0011920.pdf">https://www.miltenyibiotec.com/upload/assets/IM0011920.pdf</a> (2020)</li><li>Cell surface flow cytometry staining protocol. Miltenyi Biotec Available from: <a target="_blank" href="https://www.miltenyibiotec.com/US-en/applications/all-protocols/cell-surface-flow-cytometry-straining-protocol-pbs-bsa-1-50.html">https://www.miltenyibiotec.com/US-en/applications/all-protocols/cell-surface-flow-cytometry-straining-protocol-pbs-bsa-1-50.html</a> (2021)</li><li>Finak, G., Jiang, W., Gottardo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CytoML+for+cross-platform+cytometry+data+sharing.">CytoML for cross-platform cytometry data sharing.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 93, 1189-1196 (2018).</li><li>Tura&#231;, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+flow+cytometric+analysis+of+surface+and+intracellular+antigens+reveals+surface+molecule+markers+of+human+neuropoiesis.">Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.</a> PLoS One. 8, 1-14 (2013).</li><li>Schwarz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence+activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> Journal of Visualized Experiments: JoVE. (99), e52537 (2015).</li><li>Brummelman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=application+and+computational+analysis+of+high-dimensional+fluorescent+antibody+panels+for+single-cell+flow+cytometry.">application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry.</a> Nature Protocols. 14, 1946-1969 (2019).</li><li>Recommended controls for flow cytometry. Abcam Available from: <a target="_blank" href="https://www.abam.com/protocols/recommended-conntrols-for-flow-cytometry">https://www.abam.com/protocols/recommended-conntrols-for-flow-cytometry</a> (2021)</li><li>. Fixing cells with paraformaldehyde (PFA) for flow cytometry Available from: <a target="_blank" href="https://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf">https://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf</a> (2021)</li><li>Stewart, J. C., Villasmil, M. L., Frampton, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence+intensity+of+selected+leukocyte+surface+markers+following+fixation.">Changes in fluorescence intensity of selected leukocyte surface markers following fixation.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 71, 379-385 (2007).</li><li>Armand, E. J., Li, J., Xie, F., Luo, C., Mukamel, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing+of+brain+cell+transcriptomes+and+epigenomes.">Single-cell sequencing of brain cell transcriptomes and epigenomes.</a> Neuron. 109, 11-26 (2021).</li><li>Ho, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+single-cell+transcriptomics+in+adult+rodent+brain:+The+medium+spiny+neuron+transcriptome+revisited.">A guide to single-cell transcriptomics in adult rodent brain: The medium spiny neuron transcriptome revisited.</a> Frontiers in Cellular Neuroscience. 12, 1-16 (2018).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technologies+in+studying+brain+immune+response.">Novel technologies in studying brain immune response.</a> Oxidative Medicine and Cellular Longevity. 2021, 6694566 (2021).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+atlas+of+the+mouse+brain+at+single-cell+resolution.">DNA methylation atlas of the mouse brain at single-cell resolution.</a> bioRxiv. , (2020).</li><li>Mattei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+induces+transcriptional+and+proteotype+bias+in+brain+cell+populations.">Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations.</a> International Journal of Molecular Sciences. 21, 1-20 (2020).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+microdissected+mouse+brain+tissue+for+artifact+free+single-cell+RNA+sequencing.">Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing.</a> STAR Protocols. 2 (2), 100590 (2021).</li><li>Delmonte, O. M., Fleisher, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+Surface+markers+and+beyond.">Flow cytometry: Surface markers and beyond.</a> The Journal of Allergy and Clinical Immunology. 143, 528-537 (2019).</li><li>O'Connor, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+flow+cytometry+for+biochemical+analysis.">The relevance of flow cytometry for biochemical analysis.</a> IUBMB Life. 51, 231-239 (2001).</li><li>Menon, V., Thomas, R., Ghale, A. 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Several steps in this neural dissociation protocol require proficient technique and dexterity&#8211;perfusion, supernatant aspiration, and myelin removal. Throughout the perfusion process, the internal organs must remain intact (aside from removing the diaphragm and clipping the heart); this includes avoiding the upper chambers of the heart with the butterfly needle. While the amount of saline with heparin needed varies, transparent fluid flowing from the heart indicates the process is complete. The brain must be complet...

The hippocampus was first described by a Bolognese anatomist, Giulio Cesare Aranzio, in the 1500&#39;s1. In naming this newfound structure, Aranzio was likely inspired by its uncanny resemblance to the seahorse of the genus Hippocampus1. The hippocampus is involved in stress responses but is widely known for its role in learning and memory. More specifically, the hippocampus is responsible for the encoding and retrieval of declarative and spatial memory1.
The hippocampus, or hippocampus proper, is divided into the CA1 (cornu ammonis), CA2, and CA3 subfields1. Compared to the rest of the nervous system, the hippocampus has several unique defining characteristics, including its plasticity and potential for ongoing neurogenesis2. Neurogenesis is the process of the proliferation and differentiation of neural stem cells, followed by their integration into the pre-existing neuronal network. Neurogenesis is restricted to the subgranular zone of the dentate gyrus and subventricular zone of the lateral ventricles (and the olfactory bulbs)3. While neurogenesis is abundant in embryogenesis, it is a lifelong process3,4. As such, this discussion will focus on adult neurogenesis in the hippocampus.
The subventricular and subgranular zones are neurogenic niches containing ependymal and vascular cells, as well as immature and mature lineages of neural stem cells5. Microglia contribute to these niches as immune cells to regulate neurogenesis6. Neural progenitor cells are nonstem cell progenies of neural stem cells7. Three types of neural progenitors are present in the subventricular zone: radial glia-like type B cells, type C transit-amplifying progenitors, and type A neuroblasts3,8. The slowly dividing type B neural progenitor cells in the subventricular zone can differentiate into rapidly dividing type C cells8. Subsequently, type C cells differentiate into type A cells8. These neuroblasts migrate through the rostral migratory stream to the olfactory bulb before differentiating into interneurons or oligodendrocytes9. These olfactory bulb interneurons are key to olfactory short-term memory, and associative learning, whereas the oligodendrocytes myelinate axons of the corpus callosum9. The majority of adult neurogenesis occurs in the subgranular zone of the dentate gyrus, where radial type 1 and nonradial type 2 neural progenitors are found3. Most neural progenitor cells are destined to become dentate granule neurons and astrocytes10. Connected by gap junctions, astrocytes form networks to modulate plasticity, synaptic activity, and neuronal excitability5. As the primary excitatory neuron of the dentate gyrus, granule cells provide input from the entorhinal cortex to the CA3 region11.
Neural stem cell populations can be isolated using immunomagnetic or immunofluorescent isolation strategies12,13. Neural tissue is particularly difficult to dissociate; efforts to do so often result in samples with poor cell viability and/ or fail to produce the necessary single-cell suspension for downstream analysis. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination of techniques14,15. In a study evaluating neural dissociation methods, the viability and quality of manual mechanical dissociation by pipette trituration versus combinations of pipette trituration and digestion with various enzymes were compared15. Quality was graded based on the amount of cell clumps and DNA or subcellular debris in the prepared suspension15. Suspensions of glial tumors subjected to manual mechanical dissociation alone had significantly lower cell viability than treatments with dispase or a combination of DNase, collagenase, and hyaluronidase15. Volovitz et al. acknowledged the variation in viability and quality between the different methods and emphasized that inadequate dissociation may reduce the accuracy of downstream analysis15.
In a separate study, the authors compared over 60 different methods and combinations of dissociation of cultured neuronal cells14. These methods included eight different variations of manual mechanical dissociation by pipette trituration, a comparison of incubation with five individual enzymes at three different intervals, and various combinations of mechanical dissociation with enzymatic digestion or the combination of two enzymes14. None of the mechanical methods yielded a single-cell suspension14. Four of the single enzyme treatments, ten of the combination enzymatic treatments, and four of the combinations of mechanical dissociation with enzymatic digestion yielded a single-cell suspension14. Enzymatic digestion with TrypLE followed by Trypsin-EDTA most effectively dissociated samples14. Incidentally, samples treated with TrypLE and/or Trypsin-EDTA tended to form gelatinous clumps14. While this study was performed on cultured cells, it speaks to the shortcomings of pipette trituration or enzymatic digestion alone.
Side-by-side comparisons of manual versus automated mechanical dissociation are lacking. However, one group ran flow cytometry to compare manual and semi-automated mechanical dissociation of whole mouse brains in conjunction with commercial papain or trypsin enzymatic dissociation kits16. Processing with the dissociator more consistently yielded viable cells16. Following dissociation, the authors also isolated Prominin-1 cells, neuronal precursor cells, and microglia16. For two of the three isolated cell populations, the purity of the isolated cells was slightly higher when samples were processed with the dissociator, as compared to manually16. Rei&#223; et al. noted that person-to-person variability in pipetting technique hinders reproducibility of viable cell population yield in tissue dissociation16. The authors concluded that automated mechanical dissociation standardizes sample processing16.
The method of dissociation outlined in this manuscript is a combination of fully automated mechanical dissociation and enzymatic digestion, using solutions accompanying a commercial adult brain dissociation kit17. Unlike standard protocols, this optimized protocol reduces sample manipulation, yields a highly viable single-cell suspension, and is intended for processing minimal amounts of starting tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>02-682-003</td>
			<td>Basix, assorted color</td>
		</tr>
		<tr>
			<td>15 mL Falcon Tubes</td>
			<td>Becton Dickinson Labware Europe</td>
			<td>352009</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>25 mL Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>14-955-235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Vacuum Filter/ Storage Bottle System</td>
			<td>Corning</td>
			<td>431097</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>&#160;130-107-677</td>
			<td>Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution</td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ACSA-2-PE-Vio770, mouse, clone REA969</td>
			<td>Miltenyi Biotec</td>
			<td>130-116-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein</td>
			<td>Sigma-Aldrich</td>
			<td>M3821-100UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PSA-NCAM-PE, human, mouse and rat, Clone 2-2B</td>
			<td>Miltenyi Biotec</td>
			<td>130-117-394</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-VioBlue, mouse, Clone REA592</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-810</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-111-541</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Hot Plate Stirrer</td>
			<td>Fisher Scientific</td>
			<td>11-100-100SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD</td>
			<td></td>
			<td>(v10.7.0)</td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DPBS (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-555-25A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Fresenius Kabi</td>
			<td>504011</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L34965</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>441244-3KG</td>
			<td>Prilled, 95%</td>
		</tr>
		<tr>
			<td>Pipette tips GP LTS 20 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 250 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389277</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips RT LTS 1000 &#181;L FL 768A/8</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin Pipet-Lite XLS (2, 20, 200, 1000 &#956;L)</td>
			<td>Rainin</td>
			<td>30386597</td>
			<td></td>
		</tr>
		<tr>
			<td>RBXMO FITC XADS</td>
			<td>Fisher Scientific</td>
			<td>A16167</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Ice Bucket with Lid</td>
			<td>Fisher Scientific</td>
			<td>07-210-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Tubes with Cell Strainer Cap</td>
			<td>Falcon</td>
			<td>100-0087</td>
			<td></td>
		</tr>
		<tr>
			<td>S1 Pipet Fillers</td>
			<td>ThermoFisher Scientific</td>
			<td>9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula &#38; Probe</td>
			<td>Fine Science Tools</td>
			<td>10090-13</td>
			<td>Dissection &#38; Perfusion</td>
		</tr>
		<tr>
			<td>Surflo Winged Infusion Set 23 G x 3/4&#34;</td>
			<td>Termuno</td>
			<td>SV-23BLK</td>
			<td>Butterfly needle</td>
		</tr>
		<tr>
			<td>Test Tube Rack</td>
			<td>Fisher Scientific</td>
			<td>14-809-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Legend XTR Centrifuge</td>
			<td>ThermoFisher</td>
			<td>discontinued</td>
			<td>Or other standard table top centrifuge</td>
		</tr>
		<tr>
			<td>Variable-Flow Peristaltic Pump</td>
			<td>Fisher Scientific</td>
			<td>13-876-2</td>
			<td>Low-flow model</td>
		</tr>
		<tr>
			<td>VetFlo Starter Kit for Mice</td>
			<td>Kent Scientific</td>
			<td>VetFlo-MSEKIT</td>
			<td>Anesthesia mask, tubing, induction chamber, charcoal canisters</td>
		</tr>
		<tr>
			<td>VetFlo Vaporizer Single Channel Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>VetFlo-1210S</td>
			<td>0.2&#8211;4 LPM</td>
		</tr>
		<tr>
			<td>Vi-CELL XR Cell Viability Analyzer</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>731196</td>
			<td>Cell Counting</td>
		</tr>
		<tr>
			<td>Vi-CELL XR 4 Bags of Sample Vials</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>383721</td>
			<td>Cell Counting</td>
		</tr>
	</tbody>
</table>

combined mechanical and enzymatic dissociation of mouse brain hippocampal tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

Samples were processed with a flow cytometer at a core facility, and the resulting data were evaluated with a software package for flow analysis. Previously, compensation controls were analyzed-the live/dead stain and negative control. If multiple fluorochromes are used, fluorescence minus one (FMO) controls and single-stain controls should be prepared for each antibody. Compensation for spectral overlap for the experimental samples was calculated based on the analyzed controls. For cell population identification, a hier...

Watch this Scientific Journal Video about Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue at JoVE.com

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Successfully dissociating small quantities of neural tissue can equip labs to gain structure-specific insight into treatment efficacy, cellular function, as well as disease and treatment mechanisms of action. This neural dissociation protocol consistently yields a highly viable and actual single-cell suspension. In addition, we can process samples that are only a fraction of those that the commercial kits are intended for.Proper planning and preparation are key to a successful outcome for this technique. Running several practice rounds to familiarize yourself with the protocol would be beneficial. After anesthetizing a six-month-old female C57BL/6J mouse, pinch the lower abdomen and lift the skin using forceps.Then using scissors, cut through the fur and skin to the bottom of the ribcage. Make two diagonal incisions beginning below the ribcage and moving toward each shoulder. Then carefully resect the diaphragm and ribcage to expose the heart.After carefully removing any connective tissue around the heart, use scissors to clip the right atrium. Then turn off the isoflurane flow to the breather. Holding the heart steady with forceps and with the bevel of the butterfly needle facing up, pierce the left ventricle while keeping the needle level and parallel to the animal.Next, holding the needle in place, turn on the pump and perfuse at least 30 milliliters of the saline or heparin solution until the fluid leaving the heart is opaque and the liver and lungs pale in color. After perfusion, turn off the pump, remove the needle, and transfer the mouse to the dissection area. After decapitation, cut the fur from the back of the head up to the eyes and peel the skin back to expose the skull.Next, clip the skull between the eyes and make two cuts at the back of the skull at 10 and 2 o'clock positions. Then make one long cut along the midsagittal line of the skull to the original cut between the eyes. Next, using forceps, peel the two halves of the skull away to the sides.Then use a spatula to remove the brain and place it in a 60 millimeter glass Petri dish filled with cold DPBS and kept on ice. Using a scalpel or razor, separate each hemisphere and remove the olfactory bulbs and cerebellum. Then remove the midbrain until the hippocampus is exposed.Next, secure the brain with forceps. Then using a second set of forceps, gently tease the hippocampus out of each hemisphere. Transfer both hippocampi to a labeled 1.5 milliliter tube containing cold DPBS and place the tube on ice.Using forceps, transfer the hippocampi tissue pieces to a C tube and add 30 microliters of enzyme mix 2 into the tube. After twisting the cap and tightening until it clicks, place the tube in a dissociator and run the appropriate program. While the program is running, pre-wet a 70 micron cell strainer placed on a 50 milliliter conical tube with two milliliters of BSA buffer.After the dissociation program is complete, add four milliliters of BSA buffer to the dissociated tissue and filter the mixture through the cell strainer on the 50 milliliter conical tube. Next, add 10 milliliters of DPBS to the C tube. Then close the tube, swirl the solution gently, and filter it through the cell strainer on the 50 milliliter conical tube.Centrifuge the filtered cell suspension, discard the supernatant without disturbing the pellet and store the pellet. For debris removal, resuspend the pellet with 1, 550 microliters of cold DPBS and transfer the suspension to a labeled 50 milliliter conical tube. Then add 450 microliters of cold debris removal solution and pipette up and down.Gently overlay the cell suspension with one milliliter of cold DPBS, keeping the tip against the wall of the conical tube. Repeat the procedure until the total overlay is two milliliters. Next, centrifuge the suspension at 3, 000 times G for 10 minutes with full acceleration and full break.Aspirate the topmost layer, then sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottommost layer. Next, add two milliliters of cold DPBS and pipette up and down to mix.Then centrifuge the suspension at 1, 000 times G for 10 minutes with full acceleration and full break. After centrifugation, discard the supernatant and resuspend the pellet in one milliliter BSA buffer. After cell counting, centrifuge the remaining cell suspension and resuspend the pellet in 50 microliters of diluted live dead stain.Then transfer the sample to a labeled flow tube and incubate at room temperature for 8 to 10 minutes in the dark. After incubation, add 500 microliters of BSA buffer and repeat the centrifugation step. Then discard the supernatant, leaving a small amount of buffer in the tube.The primary gate excluded debris in the forward scatter versus side scatter plots and the dead cells were subsequently excluded. The second gate excluded cells positive for myelin basic protein. And of the remaining cells, density plots of cells positive for each fluorochrome were created.The frequency of each neuronal cell population was calculated out of the third gate. Samples processed with manual mechanical dissociation and enzymatic digestion yielded a substantially lower population of cells of interest, whereas both fresh and fixed samples processed with automated mechanical dissociation and enzymatic digestion showed a several-fold higher population of cells of interest. While the perfusion and debris removal steps can be challenging initially, maintaining a smooth and steady hand can help ensure success.

Research

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Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

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