The authors would like to thank Miss Liana Mackiewicz for technical assistance. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS and Project Grant 1031212.

1. Infection of Mice With P. berghei-ANKA
<ol>
 <li>Defrost an aliquot of cryopreserved P. berghei ANKA pRBC.</li>
 <li>Restrain a cerebral malaria-resistant BALB/c donor mouse (8-12 weeks-old) using the two-handed restraint technique. Inject mouse with 100-200 &mu;l of pRBC using a 1 ml insulin syringe (28G needle). Routinely, 1-2 donor mice are injected.</li>
 <li>On days 4-5 post-infection (p.i) remove donor mouse from cage and place it on a workstation or a disposable working pad.</li>
 <li>Gently restrain mouse by the end of the tail and using small scissors cut the tail tip (approximately 1 mm). Alternatively, a small tail puncture using a 25 G needle could be performed.</li>
 <li>Place a drop of blood from the mouse's tail vein near the end of a frosted-end microscope slide.</li>
 <li>Place a second slide (spreader) on a 45&deg; angle and back it into the drop of blood. Once the blood spreads along the edge, push the spreader-slide evenly across the microscope slide to make the blood smear. Allow the smear to air dry.</li>
 <li>Fix blood smears in 100% methanol for 30 sec and then stain slides in freshly prepared Giemsa for 10 min.</li>
 <li>Rinse blood smear with running water for 1 min, allow to air-dry and examine slide under the microscope (100x, oil immersion). Enumerate pRBC within 1,000 erythrocytes and calculate percent parasitemia. Donor mice should reach parasitemia levels between 2.5-5% before they can be bled for infection of experimental animals.</li>
 <li>Collect blood from the donor mouse by retro-orbital bleeding using a heparinized micro-hematocrit capillary tube. All experiments are performed in compliance with The Walter &amp; Eliza Hall Institute Animal Ethics Committee requirements. Depending on local Animal Ethic Committee requirements other bleeding procedures might be used. Dissolve required amount of blood in RPMI medium at a final concentration of 1X106 pRBC/0.2 ml. Consider that a normal mouse hematocrit is ~6x109 red blood cells/1 ml.</li>
 <li>Infect experimental C57BL/6 mice (8-12 weeks old) by injecting intraperitoneally (i.p) 1x106 pRBC/0.2 ml.</li>
 <li>To monitor parasitemia levels of infected mice follow steps 1.3-1.8. Parasitemia should be determined every 2-3 days starting on day 2 or 3 p.i.</li>
<li>Onset of severe malaria in C57BL/6 mice might manifest as hunched appearance, ruffed fur and low activity. Some mice may recover at this stage, but progression to loss of self-righting reflex indicates irreversible disease and animals must be euthanized. </li>
</ol>
2. Intracardial Perfusion of Mice and Brain Extraction
<ol>
 <li>Assemble a manual gravity perfusion system by securing a 500 ml reservoir to a column holder attached to the top of a 60 cm column stand. Connect plastic tubing to the bottom end of the reservoir and secure a tubing clamp to control buffer flow. Attach tubing to a 23 G needle. Fill up the reservoir with PBS (kept at 22 &deg;C), open clamp and allow buffer to run through the tubing to remove all air bubbles.</li>
 <li>Euthanize P. berghei ANKA-infected mouse by CO2 inhalation. Confirm death by the absence of pedal, orbital and respiratory responses.</li>
 <li>Pin euthanized mouse by the hind and front paws dorsally on a styrofoam dissection board contained in a plastic tray. Depending on local Animal Ethic Committee requirements anesthesia could be administered prior commencement of intracardial pefusion.</li>
 <li>Wipe ventral side with 70% ethanol.</li>
 <li>Use large scissors and forceps to open skin along the midline to expose the thoracic cavity. Fold and pin skin to the sides.</li>
 <li>Hold the sternum with fine forceps and cut the diaphragm and along both sides of sternum severing the ribs. Take care not to damage any large blood vessels.</li>
 <li>Pin the rib cage by the sternum loosely next to the head.</li>
 <li>Hold ventricles with fine forceps and carefully incise right atrium with fine scissors.</li>
 <li>Insert 23G needle attached to gravity perfusion system into left ventricle towards the ascending aorta while PBS is running. Insert only 0.5 cm of the needle tip.</li>
 <li>Perfuse mouse for 5 min or until effluent is clear.</li>
 <li>Unpin mouse and lay it on its abdomen. Wipe head with 70% ethanol.</li>
 <li>Using large scissors, make a cut just above the cervical spinal cord area.</li>
 <li>Use fine scissors to make a median caudal-rostral cut starting at the base of the skull and peel skin away to expose the skull.</li>
 <li>Holding the head, place the blades of a small scissors into each orbital cavity and make a cut between the orbits.</li>
 <li>Make a longitudinal cut along the sagittal suture and carefully peel the skull on each brain hemisphere outward.</li>
 <li>Using a spatula, gently lift the brain and place it into a 10 ml centrifuge tube containing RPMI medium.</li>
</ol>
3. Isolation of Brain-sequestered Leukocytes (BSL)
<ol>
 <li>Working in a safety cabinet, place freshly harvested brain on top of a stainless steel cell strainer (40-60 mesh size) in a Petri dish containing 3-5 ml of RPMI tissue culture medium.</li>
 <li>Cut brain tissue into small pieces.</li>
 <li>Push small pieces of brain tissue through the cell strainer using a crystal plunger.</li>
 <li>Transfer brain homogenate to a 10 ml tube and centrifuge at 250 x g for 10 min at 4 &deg;C.</li>
 <li>Dissolve pellet in 3 ml of RPMI medium, containing 0.05% Collagenase D and 2U/ml DNAse I.</li>
 <li>Rotate mixture for 30 min at room temperature. Remove cell debris by pushing the mixture through a 70 &mu;m nylon cell strainer and/or by incubating on ice for 5 min.</li>
 <li>Lay brain homogenate onto a 7 ml 30% Percoll cushion and centrifuge at 400 x g (no brakes) for 20 min at room temperature.</li>
 <li>Resuspend pellet in 1 ml of red blood cell lysis buffer and incubate on ice for 5 min to lyse adherent pRBC, which could not be removed from the brain by intracardial perfusion.</li>
 <li>Add 9 ml of RPMI medium, wash cells and centrifuge at 250 x g for 10 min at 4 &deg;C.</li>
 <li>Resuspend BSL-containing pellet in 50-100 &mu;l of RPMI medium and transfer cells to an Eppendorf tube.</li>
 <li>Dilute a 5-10 &mu;l aliquot of the cell sample 1:2 in Trypan blue for identification of viable cells.</li>
 <li>Count viable cells under the microscope using a hemocytometer. From day 6 p.i onwards, when P. berghei ANKA-infected mice display neurological signs, 20,000-100,000 BSLs can be recovered.</li>
</ol>
4. Immunophenotyping of BSL by Multicolour Flow Cytometry
<ol>
 <li>Centrifuge cells at 250 x g for 10 min at 4 &deg;C, aspirate the supernatant and resuspend pellet in 50 &mu;l of staining buffer (PBS, 1% foetal calf serum (FCS), 2 mm EDTA).</li>
 <li>Add the 1 &mu;l of purified anti-CD16/32 antibody (Fcblock).</li>
 <li>Incubate cells for 10 min on ice.</li>
 <li>Wash cells with 1 ml of Staining Buffer. Centrifuge cells at 250 x g for 10 min at 4 &deg;C and aspirate the supernatant.</li>
 <li>Resuspend cells in 50 &mu;l of Staining Buffer containing pre-determined optimal dilutions of required fluorescent antibodies, i.e. anti-CD4, anti-CD8, anti-TCR and anti-NK1.1.</li>
 <li>Incubate for 1 hr on ice.</li>
 <li>Wash cells with 1 ml of Staining Buffer. Centrifuge cells at 250 x g for 10 min at 4 &deg;C and aspirate the supernatant.</li>
 <li>Resuspend cells in 100 &mu;l of PBS.</li>
 <li>Transfer cells to a flow cytometry plastic tube.</li>
 <li>Using a flow cytometer, acquire at least 5,000-10,000 events. Appropriate unstained cell controls and fluorochrome compensation samples should be included as required.</li>
 <li>Analyze results using appropriate flow cytometry software.</li>
</ol>

<ol>
	<li>Brian de Souza, J., Riley, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+malaria:+the+contribution+of+studies+in+animal+models+to+our+understanding+of+immunopathogenesis.">Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.</a> Microbes and Infection. 4, 291-300 (2002).</li><li>Schofield, L., Grau, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+processes+in+malaria+pathogenesis.">Immunological processes in malaria pathogenesis.</a> Nature. 5, 722-735 (2005).</li><li>Miller, L. H., Baruch, D. I., Marsh, K., Doumbo, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+basis+of+malaria.">The pathogenic basis of malaria.</a> Nature. 415, 673-679 (2002).</li><li>Howe, C. L., Lafrance-Corey, R. G., Sundsbak, R. S., Lafrance, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+monocytes+damage+the+hippocampus+during+acute+picornavirus+infection+of+the+brain.">Inflammatory monocytes damage the hippocampus during acute picornavirus infection of the brain.</a> Journal of neuroinflammation. 9, 50 (2012).</li><li>LaFrance-Corey, R. G., 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=Isolation+of+Brain-infiltrating+Leukocytes.">Isolation of Brain-infiltrating Leukocytes.</a> J. Vis. Exp. (52), e2747 (2011).</li><li>Lim, S. M., Koraka, P., Osterhaus, A. D., Martina, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus:+immunity+and+pathogenesis.">West Nile virus: immunity and pathogenesis.</a> Viruses. 3, 811-828 (2011).</li><li>Belnoue, 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=On+the+pathogenic+role+of+brain-sequestered+alphabeta+CD8++T+cells+in+experimental+cerebral+malaria.">On the pathogenic role of brain-sequestered alphabeta CD8+ T cells in experimental cerebral malaria.</a> Journal of Immunology. 169, 6369-6375 (2002).</li><li>Campanella, G. S., 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=Chemokine+receptor+CXCR3+and+its+ligands+CXCL9+and+CXCL10+are+required+for+the+development+of+murine+cerebral+malaria.">Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria.</a> Proceedings of the National Academy of Science U S A. 105, 4814-4819 (2008).</li><li>Hansen, D. S., Bernard, N. J., Nie, C. Q., Schofield, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+stimulate+recruitment+of+CXCR3++T+cells+to+the+brain+during+Plasmodium+berghei-mediated+cerebral+malaria.">NK cells stimulate recruitment of CXCR3+ T cells to the brain during Plasmodium berghei-mediated cerebral malaria.</a> Journal of Immunology. 178, 5779-5788 (2007).</li><li>Amante, F. 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+role+for+natural+regulatory+T+cells+in+the+pathogenesis+of+experimental+cerebral+malaria.">A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria.</a> The American journal of pathology. 171, 548-559 (2007).</li><li>Nie, C. Q., 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=IP-10-mediated+T+cell+homing+promotes+cerebral+inflammation+over+splenic+immunity+to+malaria+infection.">IP-10-mediated T cell homing promotes cerebral inflammation over splenic immunity to malaria infection.</a> PLoS pathogens. 5, e1000369 (2009).</li><li>Franke-Fayard, B., 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=Murine+malaria+parasite+sequestration:+CD36+is+the+major+receptor,+but+cerebral+pathology+is+unlinked+to+sequestration.">Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.</a> Proceedings of the National Academy of Science U S A. 102, 11468-11473 (2005).</li><li>Nitcheu, J., 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=Perforin-dependent+brain-infiltrating+cytotoxic+CD8++T+lymphocytes+mediate+experimental+cerebral+malaria+pathogenesis.">Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis.</a> Journal of Immunololgy. 170, 2221-2228 (2003).</li><li>Lundie, R. J., 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=Blood-stage+Plasmodium+infection+induces+CD8++T+lymphocytes+to+parasite-expressed+antigens,+largely+regulated+by+CD8alpha++dendritic+cells.">Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8alpha+ dendritic cells.</a> Proceeding of the National Academy of Science U S A. 105, 14509-14514 (2008).</li></ol>

No conflicts of interest declared.

The isolation and analysis of BSL is a method that allows characterization and quantification of inflammatory cells migrating to the brain in response to tissue injury or infection in experimental mouse models. The introduction of an intracardial perfusion step for removal of the blood compartment before organ extraction and subsequent cell isolation is useful to prevent contamination of inflammatory cells with non-inflammatory circulating leukocytes. This might not be an essential requirement in neurotropic infec...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions and buffers </td>
 </tr>
 <tr>
 <td>Giemsa's azur eosin methylene blue solution</td>
 <td>Merck Millipore</td>
 <td>1.09204.0500</td>
 <td>1:10 dilution in distilled water</td>
 </tr>
 <tr>
 <td>RPMI medium</td>
 <td></td>
 <td></td>
 <td>Mouse tonicity </td>
 </tr>
 <tr>
 <td>Mouse tonicity PBS</td>
 <td></td>
 <td></td>
 <td>20 mM Sodium Phosphate, 0.149 NaCl, pH 7.3</td>
 </tr>
 <tr>
 <td>0.4%Trypan Blue </td>
 <td>Sigma Aldrich</td>
 <td>T-8154</td>
 <td>1:2 dilution</td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Worthington Biochemical</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Deoxyribonuclease (DNAse) I</td>
 <td>Sigma Aldrich</td>
 <td>D4263-5VL</td>
 <td>From bovine pancreas</td>
 </tr>
 <tr>
 <td>Percoll</td>
 <td>GE Healthcare</td>
 <td>17-0891-01</td>
 <td>30% solution in PBS </td>
 </tr>
 <tr>
 <td>Ultrapure Tris</td>
 <td>Invitrogen</td>
 <td>15505-020</td>
 <td></td>
 </tr>
 <tr>
 <td>Ammonium Chloride (NH4Cl)</td>
 <td>AnalaR</td>
 <td>10017</td>
 <td></td>
 </tr>
 <tr>
 <td>Red Cell Lysis Buffer</td>
 <td></td>
 <td></td>
 <td>17 mM Tris,14 nM NH4Cl, pH 7.2</td>
 </tr>
 <tr>
 <td>FCS</td>
 <td>Gibco</td>
 <td>1009</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA disodium salt</td>
 <td>Merck</td>
 <td>10093.5V</td>
 <td>0.1M, pH 7.2</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Antibodies and conjugates </td>
 </tr>
 <tr>
 <td>Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2</td>
 <td>BD Pharmingen</td>
 <td>553142</td>
 <td>1 &mu;l in 50 &mu;l staining buffer (0.5 mg/50 ml)</td>
 </tr>
 <tr>
 <td>FITC-anti-mouse CD4, clone H129.19</td>
 <td>BD Pharmingen</td>
 <td>553651</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-anti-mouse NK1.1, clone PK136</td>
 <td>BD Pharmingen</td>
 <td>553165</td>
 <td></td>
 </tr>
 <tr>
 <td>PerCPCy5.5-anti-mouse CD8, clone 53-6-7</td>
 <td>BD Pharmingen</td>
 <td>551162</td>
 <td></td>
 </tr>
 <tr>
 <td>APC-anti-mouse TCR-&beta;, clone H57-597</td>
 <td>BD Pharmingen</td>
 <td>553174</td>
 <td></td>
 </tr>
 <tr>
 <td>PE-anti-mouse CXCR3, clone 220803</td>
 <td>R&amp;D Systems</td>
 <td>FAB1685P</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotinylated-anti-mouse CCR5, clone C34-3448</td>
 <td>BD Pharmingen</td>
 <td>559922</td>
 <td></td>
 </tr>
 <tr>
 <td>Steptavidin-PerCP-Cy5.5</td>
 <td>BD Pharmingen</td>
 <td>551419</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment and material </td>
 </tr>
 <tr>
 <td>SuperFrost microscope slide</td>
 <td>Lomb Menzel-Gl&auml;ser</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection forceps, scissors</td>
 <td>REDA Instrumente</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>500 ml PBS reservoir</td>
 <td>Nalgene</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Rubber tubing</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>23G needle</td>
 <td>BD PrecisionGlide</td>
 <td>302008</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell dissociation kit containing metal sieve</td>
 <td>Sigma Aldrich</td>
 <td>CD-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m nylon cell strainer</td>
 <td>BD Falcon</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>GmbH Neubauer</td>
 <td>717810</td>
 <td></td>
 </tr>
 <tr>
 <td>Flow cytometry tubes</td>
 <td>BD Falcon</td>
 <td>352008</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

The results in Fig. 2 show percentages and absolute numbers of different BSL populations recovered from brains of perfused or unperfused malaria-infected and na&iuml;ve control mice. Isolated BSL were stained with PE-anti-NK1.1 and APC-anti-TCR-&beta; antibodies as indicated in the Protocol text. Consistent with previous findings 7-9, &alpha;&beta;TCR+ T cells comprised a high proportion of the BSL pool in brains of perfused malaria-infected mice (day 6 p.i). This population appeared to be significa...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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.

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. ...

isolation-analysis-brain-sequestered-leukocytes-from-plasmodium

Research

JoVE Journal

Immunology and Infection

12.7K Views.  The Walter and Eliza Hall Institute of Medical Research. 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.

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

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.

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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Isolation of Brain-infiltrating Leukocytes

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

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

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

Isolation of Leukocytes from the Human Maternal-fetal Interface

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

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Selective Harvesting of Marginating-pulmonary Leukocytes