Name: isolation of brain-infiltrating leukocytes
Uploaded: 2011-06-13T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

This work was supported by grant NS64571 from the NINDS (CLH), by an early career development award from the Mayo Clinic (CLH), and by a generous gift from Donald and Frances Herdrich (CLH). We would like to thank the Mayo Clinic Flow Cytometry Core for assistance.

1. Intracranial virus injection:
The following technique has been modified and utilized extensively by our lab and colleagues. Briefly, intracranial injection of the Daniel's strain of Theiler's murine encephalomyelitis virus (TMEV) or sham-infection (1, 10) is performed on young mice (preferably 5-6 weeks of age) to elicit brain infiltrating leukocytes (BILs). Please note that results will differ between the Daniel's strain, the BeAN strain, and the GDVII strain. For the purpose of harvesting B...

<ol>
	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <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+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

We routinely use flow cytometry to determine both the quality of the brain infiltrating cell preparation, and to distinguish different populations of immune cells 2, 9. At acute time-points, our BILs method yields high percentages of inflammatory monocytes within the CD45hi population as well as high percentages of macrophages in the CD45lo population. This indicates that a reproducible immune response within the brain can robustly be characterized by our method. 
This technique is ...

<table border="1">
	<tbody>
		<tr>
 	<th>Reagent</th>
 <th>Company</th>
 <th>Catalog number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>TMEV</td>
 <td>Howe Lab</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Daniel's strain</td>
 <td>Invitrogen</td>
 <td>21063-029</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>isoflurane</td>
 <td>Novaplus</td>
 <td>NDC 0409-3292-49</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>round-bottom Oak Ridge centrifuge tubes</td>
 <td>Nalgene</td>
 <td>3118-0030</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>11875-093</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Percoll</td>
 <td>GE Healthcare</td>
 <td>17-0891-02</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>10X PBS</td>
 <td>Roche</td>
 <td>11666789001</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Ficoll-Paque Plus</td>
 <td>GE Healthcare</td>
 <td>17-1440-02</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Trypan Blue 0.4% (w/v)</td>
 <td>Mediatech</td>
 <td>25-900-CI</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>15 ml conical tubes </td>
 <td>BD Falcon</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>7 mL glass Pyrex brand Tenbroeck tissue grinder</td>
 <td>Fisher</td>
 <td>08-414-10B</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>40 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>50 mL conical</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>bovine serum albumin</td>
 <td>Sigma</td>
 <td>A9647</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>sodium azide</td>
 <td>Sigma</td>
 <td>S8032</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>CMF-PBS</td>
 <td>Mediatech</td>
 <td>21-040-CV</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>FACS tubes</td>
 <td>BD Falcon</td>
 <td>352054</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>fetal bovine serum</td>
 <td>Sigma</td>
 <td>F4135</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>2.4G2 hybridoma</td>
 <td>ATCC</td>
 <td>HB-197</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Costar V-bottom plate</td>
 <td>Corning</td>
 <td>3894</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Allegra X-22R centrifuge or equivalent</td>
 <td>Beckman</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>96-well plate bucket and rotor</td>
 <td>Beckman</td>
 <td>S2096</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Fixed-angle rotor</td>
 <td>Beckman</td>
 <td>F0360</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>paraformaldehyde</td>
 <td>Sigma</td>
 <td>P6148</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>BD FACS Calibur</td>
 <td>BD Biosciences</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>FlowJo 7.5</td>
 <td>Tree Star, Inc.</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>CD45</td>
 <td>BD Biosciences</td>
 <td>557235</td>
 <td>clone: 30-F11</td>
 </tr> 
 <tr>
	 	<td>Gr1 (Ly6C/G)</td>
 <td>BD Biosciences</td>
 <td>553128</td>
 <td>clone: RB6-8C5</td>
 </tr>
 <tr>
 	<td>CD11b</td>
 <td>ebiosciences</td>
 <td>17-0112-83</td>
 <td>clone: M1/70</td>
 </tr> 
 <tr>
 	<td>F4/80</td>
 <td>ebiosciences</td>
 <td>17-4801-82</td>
 <td>clone: BM8</td>
 </tr> 
 </tbody>
</table>

isolation of brain-infiltrating leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

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