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 BILs, mice receive 2x105 PFU of TMEV and are infected for 24 hours.
<ol>
<li>Attach the injection needle (27 gauge, &frac14; in, Kendall), to an automatic 1 ml Hamilton syringe and set to deliver 10 &mu;l of virus.</li> 
<li>Draw up TMEV in 10 &mu;L DMEM into the syringe with careful attention to air bubbles. When appropriate, sham infected mice receive 10 &mu;l of virus-free DMEM.</li> 
<li>Just prior to injection, pour approximately 1 mL of isoflurane into a bell jar that contains a wire grid with cotton substrate underneath.</li>
<li>Place mice direclty on wire grid in the bell jar until they become lightly anesthetized, insensitive to toe-pinch and immobilized by the inhalant anesthetic, approximately 10-15 seconds. Mice should not come in direct contact with isoflurane, as substance can be irritating and caustic.</li>
<li>While under anesthesia, prepare mice for injection by quickly locating appropriate coordinates in the frontal cortex region on the skull using shaving and inking. The general location for injection is 1 mm anterior to bregma and 1 mm lateral to the sagittal suture on the right side (Figure 1). While stereotactic injection may be appropriate for some experimental situations, we have found that free-hand injection without shaving or inking is suitable for the majority of experiments. Parting the fur with the needle point along both axes is appropriate for injection.</li> 
<li>To make an injection, orient the needle perpendicular to the skull and press through the bone to approximately a 3 mm depth.</li> 
<li>Express virus into the brain by pressing the Hamilton syringe button down and waiting 2 seconds for the virus to enter the brain before withdrawing.</li>
<li>Carefully remove the needle and place the mouse in a clean, dry cage. Mice awaken from anesthesia quickly and resume normal function within minutes. Give proper attention to mice that exhibit abnormal symptoms, such as excessive bleeding from the injection site, as euthanasia of the animal may be appropriate. Proper animal handling 6 and adherence to the National Institutes of Health and Institutional Animal Care and Use Committee guidelines must be followed.</li>
</ol>
2. Brain-infiltrating leukocyte cell preparation:
<ol>
<li>Prepare round-bottom Oak Ridge centrifuge tubes which can hold a capacity of 30 ml with 10 ml RPMI 1640, 9 ml Percoll and 1 ml 10X PBS and tubes should sit at RT on the bench during the brain removal process.</li>
<li>Bring Ficoll-Paque Plus to room temperature (RT).</li>
<li>Euthanize animal by modified isoflurane overdose 5 and quickly remove the brain using a stainless steel tissue spatula and place in a 15 ml conical tube containing 5 ml RPMI on ice. Under some circumstances, PBS-perfusion may be appropriate to remove circulating peripheral blood cells from the BILs preparation. Mice should be fully and properly anesthetized prior to PBS-perfusion.</li>
<li>Transfer brain and RPMI solution to a 7 ml glass Pyrex brand Tenbroeck tissue grinder and gently homogenize with 10-15 strokes.</li>
<li>Pipet an additional 5 ml RPMI into the homogenizer and pipet up and down twice. Transfer brain homogenate (approx. 10 ml) to previously prepared round-bottom tube and gently invert 2-3 times to mix.</li>
<li>Spin brain homogenate at 7800gave for 30 min at RT in a F0360 fixed-angle rotor in a Beckman Allegra X-22R clinical centrifuge.</li>
<li>After centrifugation, remove myelin debris that has floated to the top of the gradient. Collect the leukocyte layer that floats above the red blood cell pellet (Figure 2).</li>
<li>Strain leukocyte layer through a 40 &mu;m cell strainer into a 50 ml conical. Bring solution up to 50 ml volume with RMPI.</li>
<li>Spin tubes containing diluted cell suspension in a clinical centrifuge at 1500 rpm (600gave) for 5 min at RT. </li>
<li>Aspirate the supernatant and collect the cell pellet. Resuspend the pellet in 1 ml FACS Buffer (1% bovine serum albumin, 0.02% sodium azide in calcium and magnesium-free PBS) and transfer the cell suspension to 5 ml round bottom FACS tubes.</li> 
<li>Carefully underlay suspension with 1 ml Ficoll-Paque Plus.</li> 
<li>Spin tube at 2500 rpm (1400 gave) in clinical centrifuge for 25 min at RT with no brake.</li>
<li>Remove white, fluffy layer at the interface (Figure 2) with 1 ml pipette and transfer to new 5 ml FACS tube. Wash cells with 4 ml FACS buffer.</li>
<li>Spin tube at 1500 rpm (600gave) for 5 min at RT in clinical centrifuge to pellet cells.</li>
<li>Aspirate supernatant and collect cell pellet. Resuspend the leukocyte pellet in appropriate buffer and keep on ice in new FACS tube. </li>
<li>Count cells, assess cell viability by Trypan blue exclusion and analyze by flow cytometry. In general, investigators can expect to acquire approximately 5 x 105 cells/animal.</li>
</ol>
3. Flow cytometric immunophenotyping
<ol>
<li>After isolating and counting the leukocytes, spin the cells for 3 minutes at 1500 rpm (600 gave) in a clinical centrifuge.</li>
<li>Resuspend the pellet in blocking buffer containing the following: FACS buffer, supernatant from 2.4G2 hybridoma (Fc block; anti-CD16/32) and fetal bovine serum at a ratio of 10:5:1 and incubate for 30 minutes at 4&deg;C.</li> 
<li>Prepare and add conjugated antibodies against extracellular antigens to the blocked cells at a concentration of 1:200 and incubate for 30 minutes at 4&deg;C in the dark. Stain and incubate cells in 200 &mu;l in a 96-well V-bottom plate. Appropriate controls include unstained cells and fluorochrome compensation samples, as necessary.</li>
<li>Spin the stained cells at RT for 3 minutes at 1500 rpm (600gave) in a clinical centrifuge using a rotor appropriate for 96-well plates.</li>
<li>Aspirate the supernatant and wash the cells with 200 &mu;l FACS buffer by gently pipetting up and down 3 times.</li>
<li>Repeat the above washing technique twice.</li>
<li>After washing, resuspend the cells in 2% paraformaldehyde and transfer to FACS tubes. Fixation is required for flow cytometric analysis of cells infected of Biosafety Level 2 reagents.</li>
<li>Run the fixed cells on a BD FACS Calibur following a modified flow cytometry method 8 and analyze files offline using FlowJo, WinMDI or other available flow cytometry analysis software. </li>
<li>The analysis of 50-100,000 events per sample is generally required for adequate immunophenotyping.</li>
</ol>
Directly conjugated antibodies used in this experiment:
CD45 was detected with clone 30-F11. Ly6C/G was detected with clone Gr1, RB6-8C5. CD11b was detected with clone M1/70. F4/80 was detected with clone BM8.
4. Representative Results:
We show cell phenotyping results for mouse BILs at 24 hours post infection (Figure 3). Leukocytes were stained with PerCP-conjugated anti-mouse CD45 to detect immune cells, PE-conjugated anti-mouse Ly6C/G to detect inflammatory monocytes, APC-conjugated anti-mouse CD11b and APC-conjugated anti-mouse F4/80 to detect cells of monocyte lineage. The analysis was performed with a BD FACS Calibur instrument.
<img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Figure 1"/> 
Figure 1. Anatomical localization of the injection site. The injection site is located 1 mm anterior to bregma and 1 mm lateral to the sagittal suture on the right side.
<img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Figure 2"/> 
Figure 2. Illustration of gradient separation of leukocytes and BILs. Leukocytes are initially collected directly below the myelin debris layer and above the RBC pellet in the Percoll gradient. The white, fluffy layer (BILs) at the interface is collected from the Ficoll gradient.
<img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Figure 3"/> 
Figure 3. Immunophenotype of brain-infilatrating leukocytes at 24 hr post-infection.Mouse leukocytes were isolated from the brain by differential density centrifugation of tissue homogenates prepared from animals at 24 hr after intracranial infection. Cells were stained with fluorescently-conjugated antibodies against mouse CD45, Ly6C/G, CD11b, and F4/80. Initial gating was performed by plotting CD45, a marker for immune cells, against forward scatter (FSC), an indicator of cell size (C). The CD45hi (A, D), CD45lo (B, E), and CD45neg (F, G) populations were then analyzed for relative expression levels of the monocyte and macrophage markers Ly6C/G, F4/80 (A, B, F), and CD11b (D, E, G). Ly6C/G+F4/80+CD11b+ inflammatory monocytes were found almost exclusively in the CD45hi population (A, D), consistent with the infiltration of these cells from the periphery. In contrast, Ly6C/G-F4/80loCD11b+ macrophages were found almost exclusively in the CD45lo (B, E) and CD45neg (G) populations, consistent with a resident phenotype. In numerous experiments, we have never observed CD45hi cells or Ly6C/G+F4/80+CD11b+ inflammatory monocytes in the brain of uninfected or sham-infected mice (data not shown).

<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>

No conflicts of interest declared.

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 ...

isolation-of-brain-infiltrating-leukocytes

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JoVE Journal

Immunology and Infection

19.0K ビュー.  Mayo Clinic College of Medicine. マウス脳から浸潤白血球を得るために迅速な方法が記載されている。この方法は連続Percoll勾配と白血球に富む層を選択し、精製する不連続フィコール勾配を利用しています。分離された白血球は、フローサイトメトリー測定によって特徴付けることができる。

ビデオ: Isolation of Brain-infiltrating Leukocytes

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

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 ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...