This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) grants R01-NS074387, R01-NS057711 and R21-NS091555 to M.G.C.; NIH/NINDS grants R01-NS061107, R01-NS076991, R01-NS082311 and R21-NS084275 to P.R.L.; grants from Leah&#8217;s Happy Hearts, University of Michigan Comprehensive Cancer Center awarded to M.G.C. and P.R.L; the Department of Neurosurgery, University of Michigan School of Medicine; the Michigan Institute for Clinical and Health Research, supported by NIH grant 2UL1-TR000433; the University of Michigan Cancer Biology Training Grant supported by NIH/NCI (National Cancer Institute) grant T32-CA009676; the University of Michigan Training in Clinical and Basic Neuroscience supported by NIH/NINDS grant T32-NS007222; and the University of Michigan Medical Scientist Training Program supported by NIH/NIGMS (National Institute of General Medicine Sciences) grant T32-GM007863. The authors are thankful for the academic leadership and support received from Dr. Karin Muraszko and the Department of Neurosurgery; to M. Dahlgren, D. Tomford, and S. Napolitan for superb administrative support; to M. Dzaman for outstanding technical assistance; and to Phil F. Jenkins for generous support toward the purchase of a Zeiss 3D Scanning Electron Microscope. We also acknowledge the Kuchroo laboratory at Harvard Medical School from which a modified version of the density centrifugation media-mediated strategy for isolation of brain mononuclear cells was drawn.

Note: Please review the entire protocol prior to performing experiments. Approval for the use of vertebrate animals from the appropriate institutional committee on the use and welfare of animals must be obtained prior to proceeding.
1. Preparation of Tumor Cells for Intracranial Engraftment
<ol>
	<li>Working in a class II biological safety cabinet, start by preparing GL26-Cit-NT/gal1i cell culture media by supplementing a 500 ml&#160;bottle of Dulbecco&#8217;s Modified Eagle Medium (DMEM) wi...

<ol>
	<li>Bleeker, F. E., Molenaar, R. J., Leenstra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+molecular+understanding+of+glioblastoma.">Recent advances in the molecular understanding of glioblastoma.</a> J Neurooncol. 108, 11-27 (2012).</li><li>Stupp, R., 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=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N Engl J Med. 352, 987-996 (2005).</li><li>Ostrom, Q. T., 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=CBTRUS+statistical+report:+primary+brain+and+central+nervous+system+tumors+diagnosed+in+the+United+States+in+2007-2011.">CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2007-2011.</a> Neuro Oncol. 16, iv1-63 (2014).</li><li>Louveau, A., 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=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. 523, 337-341 (2015).</li><li>Kennedy, B. C., 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=Tumor-associated+macrophages+in+glioma:+friend+or+foe?.">Tumor-associated macrophages in glioma: friend or foe?.</a> J Oncol.. 2013, 486912 (2013).</li><li>Markovic, D. S., Glass, R., Synowitz, M., Rooijen, N., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+stimulate+the+invasiveness+of+glioma+cells+by+increasing+the+activity+of+metalloprotease-2.">Microglia stimulate the invasiveness of glioma cells by increasing the activity of metalloprotease-2.</a> J Neuropathol Exp Neurol. 64, 754-762 (2005).</li><li>Zhai, H., Heppner, F. L., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/macrophages+promote+glioma+progression.">Microglia/macrophages promote glioma progression.</a> Glia. 59, 472-485 (2011).</li><li>Kmiecik, 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=Elevated+CD3++and+CD8++tumor-infiltrating+immune+cells+correlate+with+prolonged+survival+in+glioblastoma+patients+despite+integrated+immunosuppressive+mechanisms+in+the+tumor+microenvironment+and+at+the+systemic+level.">Elevated CD3+ and CD8+ tumor-infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor microenvironment and at the systemic level.</a> J Neuroimmunol. 264, 71-83 (2013).</li><li>Yang, I., Han, S. J., Sughrue, M. E., Tihan, T., Parsa, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+infiltrate+differences+in+pilocytic+astrocytoma+and+glioblastoma:+evidence+of+distinct+immunological+microenvironments+that+reflect+tumor+biology.">Immune cell infiltrate differences in pilocytic astrocytoma and glioblastoma: evidence of distinct immunological microenvironments that reflect tumor biology.</a> J Neurosurg. 115, 505-511 (2011).</li><li>Wagner, 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=Microglial/macrophage+expression+of+interleukin+10+in+human+glioblastomas.">Microglial/macrophage expression of interleukin 10 in human glioblastomas.</a> Int J Cancer. 82, 12-16 (1999).</li><li>El Andaloussi, A., Lesniak, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increase+in+CD4+CD25+FOXP3++regulatory+T+cells+in+tumor-infiltrating+lymphocytes+of+human+glioblastoma+multiforme.">An increase in CD4+CD25+FOXP3+ regulatory T cells in tumor-infiltrating lymphocytes of human glioblastoma multiforme.</a> Neuro Oncol. 8, 234-243 (2006).</li><li>Kostianovsky, A. M., Maier, L. M., Anderson, R. C., Bruce, J. N., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+regulation+of+human+monocytic/microglial+activation.">Astrocytic regulation of human monocytic/microglial activation.</a> J Immunol. 181, 5425-5432 (2008).</li><li>Curtin, J. F., 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=HMGB1+mediates+endogenous+TLR2+activation+and+brain+tumor+regression.">HMGB1 mediates endogenous TLR2 activation and brain tumor regression.</a> PLoS Med. 6, e10 (2009).</li><li>Curtin, J. F., 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=Fms-like+tyrosine+kinase+3+ligand+recruits+plasmacytoid+dendritic+cells+to+the+brain.">Fms-like tyrosine kinase 3 ligand recruits plasmacytoid dendritic cells to the brain.</a> J Immunol. 176, 3566-3577 (2006).</li><li>Mineharu, Y., 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=Engineering+the+brain+tumor+microenvironment+enhances+the+efficacy+of+dendritic+cell+vaccination:+implications+for+clinical+trial+design.">Engineering the brain tumor microenvironment enhances the efficacy of dendritic cell vaccination: implications for clinical trial design.</a> Clin Cancer Res. 17, 4705-4718 (2011).</li><li>Larocque, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+fms-like+tyrosine+kinase+3+ligand+overrides+brain+immune+privilege+and+facilitates+recognition+of+a+neo-antigen+without+causing+autoimmune+neuropathology.">Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology.</a> Proc Natl Acad Sci U S A. 107, 14443-14448 (2010).</li><li>Curtin, J. F., 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=Treg+depletion+inhibits+efficacy+of+cancer+immunotherapy:+implications+for+clinical+trials.">Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials.</a> PLoS One. 3, e1983 (2008).</li><li>Ali, 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=Combined+immunostimulation+and+conditional+cytotoxic+gene+therapy+provide+long-term+survival+in+a+large+glioma+model.">Combined immunostimulation and conditional cytotoxic gene therapy provide long-term survival in a large glioma model.</a> Cancer Res. 65, 7194-7204 (2005).</li><li>Dewey, R. A., 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=Chronic+brain+inflammation+and+persistent+herpes+simplex+virus+1+thymidine+kinase+expression+in+survivors+of+syngeneic+glioma+treated+by+adenovirus-mediated+gene+therapy:+implications+for+clinical+trials.">Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.</a> Nat Med. 5, 1256-1263 (1999).</li><li>Baker, G. 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=Natural+killer+cells+eradicate+galectin-1-deficient+glioma+in+the+absence+of+adaptive+immunity.">Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity.</a> Cancer Res. 74, 5079-5090 (2014).</li><li>Baker, G. 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=Mechanisms+of+glioma+formation:+iterative+perivascular+glioma+growth+and+invasion+leads+to+tumor+progression,+VEGF-independent+vascularization,+and+resistance+to+antiangiogenic+therapy.">Mechanisms of glioma formation: iterative perivascular glioma growth and invasion leads to tumor progression, VEGF-independent vascularization, and resistance to antiangiogenic therapy.</a> Neoplasia. 16, 543-561 (2014).</li><li>Ormerod, M. G., Novo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow cytometry : a basic introduction. , (2008).</li><li>Stirling, D. P., Yong, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+inflammatory+response+after+murine+spinal+cord+injury+revealed+by+flow+cytometry.">Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry.</a> J Neurosci Res. 86, 1944-1958 (2008).</li><li>Hirai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+and+phenotype+of+activated+microglia/macrophages+within+the+spinal+cord+of+the+hyperostotic+mouse+(twy/twy)+changes+in+response+to+chronic+progressive+spinal+cord+compression:+implications+for+human+cervical+compressive+myelopathy.">The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinal cord compression: implications for human cervical compressive myelopathy.</a> PLoS One. 8, e64528 (2013).</li><li>Fleming, T. J., Fleming, M. L., Malek, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+Ly-6G+on+myeloid+lineage+cells+in+mouse+bone+marrow.+RB6-8C5+mAb+to+granulocyte-differentiation+antigen+(Gr-1)+detects+members+of+the+Ly-6+family.">Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.</a> J Immunol. 151, 2399-2408 (1993).</li><li>Wiesner, S. M., 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=De+novo+induction+of+genetically+engineered+brain+tumors+in+mice+using+plasmid+DNA.">De novo induction of genetically engineered brain tumors in mice using plasmid DNA.</a> Cancer Res. 69, 431-439 (2009).</li><li>Holland, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+activation+of+Ras+and+Akt+in+neural+progenitors+induces+glioblastoma+formation+in+mice.">Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice.</a> Nat Genet. 25, 55-57 (2000).</li><li>Lynes, 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=Lentiviral-induced+high-grade+gliomas+in+rats:+the+effects+of+PDGFB,+HRAS-G12V,+AKT,+and+IDH1-R132H.">Lentiviral-induced high-grade gliomas in rats: the effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H.</a> Neurotherapeutics. 11, 623-635 (2014).</li><li>de Vries, N. A., 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=Rapid+and+robust+transgenic+high-grade+glioma+mouse+models+for+therapy+intervention+studies.">Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies.</a> Clin Cancer Res. 16, 3431-3441 (2010).</li><li>Uhrbom, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ink4a-Arf+loss+cooperates+with+KRas+activation+in+astrocytes+and+neural+progenitors+to+generate+glioblastomas+of+various+morphologies+depending+on+activated+Akt.">Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt.</a> Cancer Res. 62, 5551-5558 (2002).</li><li>Marumoto, T., 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=Development+of+a+novel+mouse+glioma+model+using+lentiviral+vectors.">Development of a novel mouse glioma model using lentiviral vectors.</a> Nat Med. 15, 110-116 (2009).</li><li>Assanah, M., 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=Glial+progenitors+in+adult+white+matter+are+driven+to+form+malignant+gliomas+by+platelet-derived+growth+factor-expressing+retroviruses.">Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses.</a> J Neurosci. 26, 6781-6790 (2006).</li></ol>

This protocol describes a robust and reproducible method for the isolation and flow cytometric analysis of PBMCs that have infiltrated the early mouse brain tumor microenvironment. Glioma cell suspensions are generated at a concentration specified by the experimentalist that are stereotactically engrafted into the striatum of the mouse brain. Mice are then euthanized at predetermined time points specified by the experimental design and their brains are harvested and processed to isolate glioma-infiltrating PBMCs which ar...

Gliomas are a class of neuroepithelial brain cancers arising from transformed glia within the central nervous system (CNS). Of all the gliomas, World Health Organization (WHO) grade IV glioma, or glioblastoma (GBM), is the most common and lethal1. GBM is highly refractory to the current standard-of-care which consists of tumor resection to the extent possible followed by radiation plus concomitant and adjuvant chemotherapy with temozolomide2. These deadly cancers carry a dismal prognosis of only 15-18 months of survival from the time of initial diagnosis with only 5% of patients surviving the disease after 5 years3.
The presence of the blood brain barrier (BBB), lack of professional antigen presenting cells (APCs), and the previously unidentified existence of bona fide lymphatic structures within the brain4 have led to the notion of GBM as immune privileged. However, numerous studies now show that these brain cancers indeed engender the recruitment of peripheral immune cells that are predominantly myeloid in origin which include monocytes, macrophages, and myeloid-derived suppressor cells (MDSCs)5. GBM also influences the activity of brain-resident microglia to become pro-tumorigenic6,7. Lymphoid cells such as CD8+ T cells8 and CD56+&#160;natural killer&#160;cells9 are also present within the tumor microenvironment, but in much fewer numbers, a fact thought to be due to immunosuppressive function instigated by glioma-derived factors on tumor associated macrophages (TAMs)10. CD4+ T cells are also present in GBM, but much of this population also expresses CD25 and FoxP3, makers of immunosuppressive T regulatory (Treg) cells11. The overall immunosuppressive state of GBM culminates in the promotion of immunologic escape and tumor progression12.
A better understanding of the mechanisms of GBM immunosuppression is critical to the development of effective immunotherapeutic strategies designed to stimulate the immune system against the tumor. Over the last 15 years our lab has worked to overcome the mechanisms of brain tumor immunosuppresson in order to develop efficacious new anti-GBM immunotherapeutics13-19. The culmination of this work has now led to a clinical trial designed to evaluate a combined cytotoxic and immune-stimulatory therapeutic for patients with newly diagnosed GBM (ClinicalTrials.gov Identifier: NCT01811992).
Our most recent work shows that mouse GL26 and rat CNS-1 GBM cells block anti-tumor NK cell immune surveillance by producing large amounts of the &#946;-galactoside-binding lectin galectin-1 (gal-1)20. This was demonstrated by suppressing the expression of gal-1 in glioma cells using shRNA-mediated gene knockdown. In vitro experiments showed that gal-1-deficient glioma cells proliferated normally in culture, yet underwent rapid rejection soon after intracranial engraftment into syngeneic C57BL/6J or RAG1-/- mice, thus establishing the independence of T- or B- cells on this form of tumor rejection. NK cell immunodepletion with anti-asialo GM1 anti-serum or monoclonal NK1.1 antibodies led to the complete restoration of intracranial gal-1-deficient glioma growth, establishing the role of NK cells in gal-1-deficient glioma rejection. We now show that immunodepletion of Gr-1+/CD11b+ myeloid cells is sufficient to prevent gal-1-deficient glioma rejection despite the presence of NK cells, thus revealing a indispensible auxiliary role for myeloid cells in the aiding of NK-mediated gal-1-deficient tumor lysis (unpublished data). This unexpected result has led us to develop a comprehensive protocol for the isolation and analysis of peripheral blood mononuclear cells (PBMCs) that infiltrate the brain tumor microenvironment soon after intracranial engraftment so that we may better characterize the immune infiltration events that predicate gal-1-deficient glioma rejection.
The method is demonstrated here by using mouse GL26 glioma cells that constitutively express mCitrine fluorescent protein, called GL26-Cit, which permit direct tumor cell visualization by fluorescence microscopy21. These cells are stereotactically engrafted into the brain of syngeneic C57BL/6J mice and are allowed to grow for 24, 48, or 72 hr prior to mouse euthanasia. Glioma-infiltrating PBMCs are then isolated and immunolabeled using anti -CD45, -Gr-1, -CD11b and -NK1.1 cell surface antibodies together with intracellular immunolabeling for granzyme B (GzmB). This specific combination of antibodies allows for the identification of tumor-infiltrating Gr-1+/CD11b+ myeloid cells and NK1.1+, NK cells, cell types we&#160;have been implicated in gal-1-deficient tumor rejection. The immune infiltration profile of gal-1-deficient GL26-Cit glioma, referred to here as GL26-Cit-gal1i, is then compared to that of gliomas expressing normal levels of gal-1 called GL26-Cit-NT that contain a non-targeting control shRNA hairpin. The protocol begins with a description on how to culture GL26-Cit glioma cells in vitro, which is followed by an explanation on how to orthotopically engraft these cells into the striatum of syngeneic C57BL/6J mice. It then proceeds to enumerate the steps involved in the isolation and immunolabeling of glioma-infiltrating PBMCs for flow cytometric analysis. The protocol concludes with an explanation of standard data analysis and graphical representation.
The demonstration reveals that both Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells preferentially accumulate within the gal-1-deficient brain tumor microenvironment within 48 hr of tumor implantation, a result which helps explain why these tumors rapidly undergo complete tumor lysis approximately 1 week post-tumor engraftment20. The method is easily adaptable to a number of different in vivo experimental designs in which temporal data on immune infiltration into the brain is required. A single experimentalist can perform the protocol from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in about 4-6 hr depending on the number of samples to be analyzed. The method may also be combined with experiments aimed to characterize the profile of circulating PBMCs in tumor bearing mice for comparison with those that infiltrate the brain so to identify immunosuppression phenotypes specifically induced by the tumor microenvironment. Application of this and similar methods should facilitate a better understanding of the factors involved in the trafficking of peripheral immune cells into the brain tumor microenvironment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modification of Eagles Medium</td>
			<td>Gibco</td>
			<td>12430-054</td>
			<td>with 4.5g/L D-glucose, L-glutamine, 25mM HEPES and phenol red&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-buffered Saline&#160;</td>
			<td>Gibco</td>
			<td>14190-144&#160;</td>
			<td>without calcium chloride or Magnesium chloride</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Omega Scientific Inc.</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10,000U/ml Penicillin; 10,000&#956;g/ml Streptomycin&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200mM</td>
		</tr>
		<tr>
			<td>G418 sulfate&#160;</td>
			<td>Omega Scientific Inc.</td>
			<td>GN-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>from Streptomyces alboniger</td>
		</tr>
		<tr>
			<td>HyClone HyQTase non-mammalian trypsin alternative&#160;</td>
			<td>Thermo Scientific</td>
			<td>SV30030.01</td>
			<td>in DPBS with EDTA&#160;</td>
		</tr>
		<tr>
			<td>Phase contrast hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>0.1mm deep</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 0.9% NaCl injection, USP</td>
			<td>Hospira</td>
			<td>NDC: 0409-4888</td>
			<td>10ml vials</td>
		</tr>
		<tr>
			<td>0.6ml conical polypropylenemi microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole hydrochloride injection</td>
			<td>Orion Pharma</td>
			<td>NDC: 52483-6298</td>
			<td>5mg/ml solution</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Fort Dodge</td>
			<td>NDC: 0409-2051</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride injection</td>
			<td>Zoetis</td>
			<td>NDC: 54771-2805</td>
			<td>0.5mg/ml solution</td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride injection</td>
			<td>Lloyd</td>
			<td>NDC: 61311-481</td>
			<td>100mg/ml solution</td>
		</tr>
		<tr>
			<td>Carprofen injection</td>
			<td>Pfizer</td>
			<td>NDC: 61106-8501</td>
			<td>50mg/ml solution</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride injection</td>
			<td>Reckitt Benckiser</td>
			<td>NDC: 12496-0757</td>
			<td>0.3mg/ml ampuls</td>
		</tr>
		<tr>
			<td>1ml tuberculin syringes&#160;</td>
			<td>Covidien</td>
			<td>8881501400</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x &#189; (0.45mm x 13mm) syringe needles&#160;</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical clippers</td>
			<td>3M</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Providone-iodine solution</td>
			<td>Aplicare</td>
			<td>82-217</td>
			<td>NDC: 52380-1905</td>
		</tr>
		<tr>
			<td>Sterile petrolatum ophthalmic ointment&#160;</td>
			<td>Dechra</td>
			<td>NDC: 17033-211</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol prep pads</td>
			<td>Kendall</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td>non-woven, 4&#34; x 4&#34;, 4-ply&#160;</td>
		</tr>
		<tr>
			<td>1.7ml conical polypropylene microtubes</td>
			<td>Genesee Scientific</td>
			<td>22-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse stereotactic frame</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical lamp</td>
			<td>Philips Burton</td>
			<td>CS316W</td>
			<td>Coolspot II Variable Spotlight</td>
		</tr>
		<tr>
			<td>Curved dissecting forceps</td>
			<td>Ted Pella Inc.</td>
			<td>5431</td>
			<td></td>
		</tr>
		<tr>
			<td>Colibri retractors&#160;</td>
			<td>FST</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless precision power drill</td>
			<td>Dremel&#160;</td>
			<td>1100-01</td>
			<td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td>
		</tr>
		<tr>
			<td>Engraving Cutter</td>
			<td>Dremel</td>
			<td>105</td>
			<td>1/32&#34; or 0.8 mm bit diameter</td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>75</td>
			<td>Hamilton Microliter&#174; Syringes 700 series; 5&#956;l volume</td>
		</tr>
		<tr>
			<td>Microliter syringe needles</td>
			<td>Hamilton</td>
			<td>7762-06</td>
			<td>33G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 black monofilament nylon sutures&#160;</td>
			<td>Ethicon</td>
			<td>1663</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bar</td>
			<td>VWR</td>
			<td>74950-296</td>
			<td>7.9mm diameter x 50mm length (5/16&#34; diameter x 2&#34; length)</td>
		</tr>
		<tr>
			<td>1L glass screw-cap storage bottle</td>
			<td>Corning</td>
			<td>1395</td>
			<td>Type 1, Class A borosilicate glass</td>
		</tr>
		<tr>
			<td>Heparin sodium&#160;</td>
			<td>Sagent</td>
			<td>NDC: 25021-400</td>
			<td>1,000U/ml solution</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole Parmer Instruments Group</td>
			<td>77200-60</td>
			<td>Master Flex Easy Load II console drive</td>
		</tr>
		<tr>
			<td>compressed carbogen (95% O2 / 5% CO2)</td>
			<td>available from local vendor</td>
			<td>N/A</td>
			<td>A-type, large cylinder&#160;</td>
		</tr>
		<tr>
			<td>20G x 1 &#189;&#34; aluminum hub blunt needles</td>
			<td>Kendall</td>
			<td>8881202363</td>
			<td>0.9mm x 38.1mm</td>
		</tr>
		<tr>
			<td>Polyurethane ice bucket</td>
			<td>Fisherbrand</td>
			<td>02-591-45</td>
			<td>Capacity: 0.152 oz. (4.5L)</td>
		</tr>
		<tr>
			<td>Collagenase (Type I-S)</td>
			<td>Sigma Aldrich</td>
			<td>C1639</td>
			<td>from Clostridium histolyticum&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS002007</td>
			<td>&#62;2,000 Kunitz units per mg dry weight</td>
		</tr>
		<tr>
			<td>Antistatic polystyrene hexagonal weighing dishes</td>
			<td>Ted Pella Inc.</td>
			<td>20157-3</td>
			<td>top I.D. 115mm, base I.D. 85mm, 203ml volume&#160;</td>
		</tr>
		<tr>
			<td>Stainless steel single edged razor blades&#160;</td>
			<td>Garvey</td>
			<td>40475</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone rongeurs</td>
			<td>FST</td>
			<td>16001-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FST</td>
			<td>13014-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Large dissection scissors</td>
			<td>Ted Pella Inc.</td>
			<td>1316</td>
			<td></td>
		</tr>
		<tr>
			<td>Small dissection scissors</td>
			<td>FST</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Ted Pella Inc.</td>
			<td>13250</td>
			<td></td>
		</tr>
		<tr>
			<td>7ml glass Dounce tissue grinder</td>
			<td>Kontes</td>
			<td>KT885300-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll Density Centrifugation Media&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Genesee Scientific</td>
			<td>12-104</td>
			<td>single use; sterile</td>
		</tr>
		<tr>
			<td>70&#956;m sterile nylon mesh cell strainers&#160;</td>
			<td>Fisherbrand</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td>
			<td>Biolegend</td>
			<td>103128</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td>
			<td>eBiosciences&#160;</td>
			<td>17-5941-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td>
			<td>BD Pharmigen</td>
			<td>553128</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&#160;</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 700-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400628</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG2a, &#954; (clone: eBM2a) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>17-4724</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated rat IgG2b, &#954; (clone: eB149/10H5) isotype control&#160;</td>
			<td>eBioscience</td>
			<td>12-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5-conjugated rat IgG2b, &#954; (clone: RTK4530) isotype control</td>
			<td>Biolegend</td>
			<td>400632</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&#160;</td>
			<td>Biolegend</td>
			<td>515403</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Blue-conjugated mouse IgG1, &#954; (Clone: MOPC-21) isotype control&#160;</td>
			<td>Biolegend</td>
			<td>400151</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-103</td>
			<td>conical bottom&#160;</td>
		</tr>
		<tr>
			<td>50ml polypropylene centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td>conical bottom</td>
		</tr>
		<tr>
			<td>12x75mm round-bottom polypropylene FACS tubes&#160;</td>
			<td>Fisherbrand</td>
			<td>14-956-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Special Order Research Product flow cytometer/cell sorter</td>
			<td>BD&#160;</td>
			<td>650033</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG603</td>
			<td>model: SterilGARD III Advance</td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

The following gating strategy is used for a typical experiment: FSC-A vs. SSC-A &#8594; SSC-H vs. SSC-W &#8594; FSC-H vs. FSC-W &#8594; CD45 vs. count &#8594; Gr-1 vs. CD11b &#8594; NK1.1 vs. count. Gates placed on Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells are then stratified based on GzmB expression (Figure 5A). Backgating those cells identified as NK1.1+ NK cells and Gr-1+/CD11b+ myeloid cells in our experiments onto FSC-A vs. SSC-A...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Research

JoVE Journal

Immunology and Infection

15.6K 조회수.  University of Michigan.  초기 뇌 종양 미세 환경에 진입 면역 세포의 수와 활성 상태에서 시간에 따른 양적 데이터를 산출 교종 함침 말초 혈액 단핵 세포의 분리 및 유세포 분석을위한 간단한 방법이 여기에서 제시 하였다. 