Name: isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells
Uploaded: 2015-11-28T15:00:00.000+00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

이 작품은 신경 질환 및 뇌졸중 (NIH / NINDS) MGC에 R01-NS074387, R01-NS057711 및 R21-NS091555 부여의 / 국립 연구소 건강의 국립 연구소에 의해 지원되었다; NIH / NINDS는 PRL에 R01-NS061107, R01-NS076991, R01-NS082311 및 R21-NS084275을 부여; 레아의 행복한 마음에서 보조금, MGC 및 PRL에게 수여 미시간 종합 암 센터의 대학; 신경 외과, 의학 미시간 대학의 대학의 부; NIH 보조금 2UL1-TR000433 지원하는 임상에 대한 미시간 연구소 및 건강 연구,; NIH / NCI (국립 암 연구소) 부여 T32-CA009676에 의해 지원되는 미시간 대학 암 생물학 교육 보조금; NIH / NINDS 지원하는 임상과 기초 신경 과학 미시간 교육 대학은 T32-NS007222을 부여; 그리고 NIH / NIGMS (일반 의학 과학의 국립 연구소)에 의해 지원 미시간 의료 과학자 교육 프로그램의 대학은 T32-GM007863을 부여합니다. 저자박사 카린 Muraszko 및 신경 외과의 부서에서받은 교육 리더십과 지원에 감사 재; M. 달 그렌, D. Tomford,과 최상의 행정 지원에 대한 S. 나폴리탄에; 뛰어난 기술 지원 M. Dzaman에; 그리고 자이스 3D 스캐닝 전자 현미경의 구입을 향해 관대 한 지원을위한 필 F. 젠킨스에. 우리는 또한 뇌 단핵 세포의 분리를위한 밀도 원심 분리 미디어 매개 전략의 수정 된 버전이 그려진하는 하버드 의과 대학에서 Kuchroo 실험실을 인정합니다.

<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>

저자가 공개하는 게 없다.

이 프로토콜은 초기 마우스 뇌 종양 미세 환경에 침투 한 한 PBMC의 분리 및 유세포 분석을위한 견고하고 재생 가능한 방법을 설명한다. 신경 교종 세포 현탁액 정위 마우스 뇌의 선조체에 접목 된 실험자에 의해 지정된 농도로 생성된다. 마우스는 다음 실험 디자인에 의해 지정된 소정의 시점에서 안락사 그들의 뇌를 수거하고, 형광 접합 된 일차 항체의 조합으로되어 immunolabeled 교종 함침 한 PBMC?...

신경 교종은 중추 신경계 (CNS)에서 발생하는 변형 신경교 neuroepithelial 뇌암의 클래스이다. 모든 신경 교종의, 세계 보건기구 (WHO) 등급 IV 신경 교종, 또는 아교 모세포종 (GBM), 가장 일반적인 1 치명적이다. GBM은 테모 졸로 마이드 2 방사선 플러스 수반과 보조 화학 요법 다음에 가능한 범위에 종양 절제술로 구성되어 현재의 표준 진료에 매우 내화입니다. 이 치명적인 암 5 년 3 후에 질병을 생존 환자의 5 %와 초기 진단의 시간에서 생존의 15-18개월의 실력 예후를 수행한다. 혈액 뇌 장벽 (BBB)의 존재는, 전문적인 항원 제시 세포의 부족 (장갑차) 및 뇌 내에서 4 선의 림프 구조의 존재는 이전 미확인 특권 면역 GBM의 개념을 이끌어왔다. 그러나, 많은 연구가 지금 SHO이러한 뇌 암은 참으로 단핵구, 대 식세포 및 골수 유래 억제 세포 (MDSCs) (5)를 포함 기원에 주로 골수 있습니다 주변 면역 세포의 채용을 발생시킬 것을 승. GBM은 6,7 프로 종양 될 뇌 상주 미세 아교 세포의 활동에 영향을 미친다. 이러한 CD8 + T 세포 (8)과 CD56 + 자연 살해 세포 (9) 등의 림프 세포는 종양 미세 환경 내 존재하지만 훨씬 적은 숫자 생각 사실 (인해 종양 연관된 대 식세포에 신경 교종 - 유래 요인에 의해 유발 면역 기능 할 TAMS) 10. CD4 + T 세포는 GBM도 존재하지만이 인구의 대부분은 CD25 및 Foxp3를, 면역 T 규제 (T의 REG) 셀 (11)의 업체를 표현한다. GBM의 전반적인 면역 억제 상태는 면역 탈출 및 종양 진행 (12)의 증진에 절정. 13-19을 개발하기 위해 뇌 종양 immunosuppresson의 메커니즘을 극복하기 위해 노력하고있다. 이 작품의 절정은 이제 새로 진단 GBM (: NCT01811992 ClinicalTrials.gov 식별자) 환자 결합 된 세포 독성 및 면역 자극 치료를 평가하기위한 임상 시험을 주도하고있다. 가장 최근의 연구는 마우스 GL26 쥐 CNS -1- GBM 셀 β - 갈 락토 시드 - 결합 렉틴 ​​galectin-1 (GAL-1) ~ (20)의 대량 생산함으로써 항암 NK 세포의 면역 감시를 차단하는 것으로 나타났다. 이것은 shRNA를 매개 유전자 녹다운을 사용하여 신경 교종 세포의 GAL-1의 발현을 억제함으로써 입증되었다. 시험 관내 experimenTS는 GAL-1 결핍 신경 교종 세포 배양에 정상적으로 증식 것으로 나타났다, 아직 동계 C57BL / 6J 또는 RAG1에 두개 내 생착 후 곧 빠른 거절을 시행 - / - 마우스, 따라서 이러한 형태에 T- 또는 B- 세포의 독립성을 확립 종양 제거. 안티 아시 알로 GM 1 항혈청 또는 GAL-1 결핍 신경 교종 제거에서 NK 세포의 역할을 설정하는 두개 GAL-1 결핍 교종 성장의 완전한 회복되었다 모노클 NK1.1 항체와 NK 세포 immunodepletion. 우리는 지금 GR-1 + /는 CD11b + 골수 세포의 immunodepletion 표시하는 따라서 NK 매개 GAL의 돕는데 골수 세포를위한 필수 불가결 한 보조 역할을 드러내는, NK 세포의 존재에도 불구하고 GAL-1 결핍 신경 교종 제거를 방지하기에 충분한 -1 결핍 종양 용해 (게시되지 않은 데이터). 이는 예기치 않은 결과는 말초 혈 단핵 세포의 분리 및 분석 (한 PBMC)를위한 광범위한 프로토콜을 개발할 주도했음을우리는 더 나은 GAL-1 결핍 신경 교종 거부 조건부 면역 침투 이벤트를 특성화 할 수 있도록 즉시 두개 내 생착 후 뇌 종양 미세 환경에 침투. 방법은 GL26-CIT를 불리는 구조적 익스프레스 mCitrine 형광 단백질은, 형광 현미경 (21)에 의해 직접 종양 세포의 가시화를 허용 GL26 마우스의 신경 교종 세포를 사용하여 여기에 설명된다. 이 세포들은 정위 동계 C57BL / 6J 마우스의 뇌에 접목하고, 이전에 마우스 안락사에 24, 48, 또는 72 시간 동안 성장을 사용할 수 있습니다. 신경 교종-침투 PBMC를 다음 항 -CD45를 사용하여 격리 및 immunolabeled되어, -GR-1, -CD11b과 그랜 자임 B의 세포 내 immunolabeling와 함께 -NK1.1 세포 표면 항체 (GzmB). 항체의 이러한 특정 조합이 우리가 B가 종양 침윤 GR-1 + / + CD11b를 골수 세포의 식별 및 NK1.1 +, NK 세포, 세포 유형을 허용EEN는 GAL-1 결핍 종양 제거에 관여. GAL-1 결핍 GL26-CIT의 신경 교종의 면역 침투 프로파일, GL26-CIT의-gal1i 여기에 언급하고 비를 포함 GAL-1 GL26-CIT의-NT 호출의 정상 수준을 발현하는 신경 교종의 것과 비교된다 제어 shRNA를 머리핀을 대상으로. 프로토콜 동소 동계 C57BL / 6J 마우스의 선조체로 이들 세포를 접목하는 방법에 대한 설명이 뒤 따른다 시험 관내 배양 방법 GL26-CIT의 신경 교종 세포에 대한 설명으로 시작한다. 그러므로 분리 및 유세포 분석 교종 함침 된 PBMC의 immunolabeling 관련된 단계를 열거 진행한다. 표준 프로토콜은 데이터 분석 및 그래픽 표현의 설명으로 종결. 데모는 밝혀 그 모두 GR-1 + /는 CD11b + 골수 세포와 NK1.1 + NK 세포 우선적으로 48 내 여자-1 결핍 뇌 종양 미세 환경 내에서 축적종양 주입,이 종양이 빠른 속도로 완전한 종양 용해 약 일주 후 종양 생착 (20)를 받아야하는 이유를 설명하는 데 도움이되는 결과의 시간. 상기 방법은 뇌에 면역 침윤 시간에 데이터를 필요로하는 생체 내 다양한 실험 디자인에 용이하게 적응할 수있다. 실험자들은 단일 분석 될 샘플의 수에 따라 약 4-6 시간에 신경 교종 함침 된 PBMC의 세포 계측 분석을 흐르게 뇌 적출로부터 프로토콜을 수행 할 수있다. 방법은 또한 특히 종양 미세 환경에 의해 유도 된 면역 표현형을 식별하도록 뇌 침투 것들과의 비교를 위해 종양 함유 마우스에서 PBMC를 순환 프로파일을 특성화하기 위해 실험 목적과 결합 될 수있다. 이것과 유사한 방법을 적용 뇌 종양 미세 환경 내로 말초 면역 세포의 거래에 관련된 요인의 더 나은 이해를 용이하게한다.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modification of Eagles Medium</td><td>Gibco</td><td>12430-054</td><td>with 4.5g/L D-glucose, L-glutamine, 25 mM HEPES and phenol red&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline&amp;nbsp;</td><td>Gibco</td><td>14190-144&amp;nbsp;</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&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td>10,000 U/ml Penicillin; 10,000 &amp;mu;g/ml Streptomycin&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td>200 mM</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Omega Scientific Inc.</td><td>GN-04</td><td></td></tr><tr><td>Puromycin dihydrochloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8833</td><td>from Streptomyces alboniger</td></tr><tr><td>HyClone HyQTase non-mammalian trypsin alternative&amp;nbsp;</td><td>Thermo Scientific</td><td>SV30030.01</td><td>in DPBS with EDTA&amp;nbsp;</td></tr><tr><td>Phase contrast hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>0.1 mm 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>10 ml vials</td></tr><tr><td>0.6 ml 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>5 mg/ml solution</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Fort Dodge</td><td>NDC: 0409-2051</td><td>100 mg/ml solution</td></tr><tr><td>Dexmedetomidine hydrochloride injection</td><td>Zoetis</td><td>NDC: 54771-2805</td><td>0.5 mg/ml solution</td></tr><tr><td>Xylazine hydrochloride injection</td><td>Lloyd</td><td>NDC: 61311-481</td><td>100 mg/ml solution</td></tr><tr><td>Carprofen injection</td><td>Pfizer</td><td>NDC: 61106-8501</td><td>50 mg/ml solution</td></tr><tr><td>Buprenorphine hydrochloride injection</td><td>Reckitt Benckiser</td><td>NDC: 12496-0757</td><td>0.3 mg/ml ampuls</td></tr><tr><td>1 ml tuberculin syringes&amp;nbsp;</td><td>Covidien</td><td>8881501400</td><td></td></tr><tr><td>26G x &amp;frac12; (0.45 mm x 13 mm) syringe needles&amp;nbsp;</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&amp;nbsp;</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&quot; x 4&quot;, 4-ply&amp;nbsp;</td></tr><tr><td>1.7 ml 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&amp;nbsp;</td><td>FST</td><td>17000-03</td><td></td></tr><tr><td>Cordless precision power drill</td><td>Dremel&amp;nbsp;</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&quot; or 0.8 mm bit diameter</td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>75</td><td>Hamilton Microliter Syringes 700 series; 5 &amp;mu;l volume</td></tr><tr><td>Microliter syringe needles</td><td>Hamilton</td><td>7762-06</td><td>33 G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td></tr><tr><td>Ethilon 3-0 black monofilament nylon sutures&amp;nbsp;</td><td>Ethicon</td><td>1663</td><td></td></tr><tr><td>Magnetic stir bar</td><td>VWR</td><td>74950-296</td><td>7.9 mm diameter x 50 mm length (5/16&quot; diameter x 2&quot; length)</td></tr><tr><td>1 L 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&amp;nbsp;</td><td>Sagent</td><td>NDC: 25021-400</td><td>1,000 U/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% O&lt;sub&gt;2 &lt;/sub&gt;/ 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>available from local vendor</td><td>N/A</td><td>A-type, large cylinder&amp;nbsp;</td></tr><tr><td>20 G x 1 &amp;frac12;&quot; aluminum hub blunt needles</td><td>Kendall</td><td>8881202363</td><td>0.9 mm x 38.1 mm</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&amp;nbsp;</td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical Corp.</td><td>LS002007</td><td>&gt;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. 115 mm, base I.D. 85 mm, 203 ml volume&amp;nbsp;</td></tr><tr><td>Stainless steel single edged razor blades&amp;nbsp;</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>7 ml glass Dounce tissue grinder</td><td>Kontes</td><td>KT885300-0007</td><td></td></tr><tr><td>Percoll Density Centrifugation Media&amp;nbsp;</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 &amp;mu;m sterile nylon mesh cell strainers&amp;nbsp;</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&amp;nbsp;</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)&amp;nbsp;</td><td>Biolegend</td><td>101228</td><td></td></tr><tr><td>Alexa Fluor 700-conjugated rat IgG2b, &amp;kappa; (clone: RTK4530) isotype control&amp;nbsp;</td><td>Biolegend</td><td>400628</td><td></td></tr><tr><td>APC-conjugated mouse IgG2a, &amp;kappa; (clone: eBM2a) isotype control&amp;nbsp;</td><td>eBioscience</td><td>17-4724</td><td></td></tr><tr><td>PE-conjugated rat IgG2b, &amp;kappa; (clone: eB149/10H5) isotype control&amp;nbsp;</td><td>eBioscience</td><td>12-4031-82</td><td></td></tr><tr><td>PerCP/Cy5.5-conjugated rat IgG2b, &amp;kappa; (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)&amp;nbsp;</td><td>Biolegend</td><td>515403</td><td></td></tr><tr><td>Pacific Blue-conjugated mouse IgG1, &amp;kappa; (Clone: MOPC-21) isotype control&amp;nbsp;</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>15 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-103</td><td>conical bottom&amp;nbsp;</td></tr><tr><td>50 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-108</td><td>conical bottom</td></tr><tr><td>12 mm x 75 mm round-bottom polypropylene FACS tubes&amp;nbsp;</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&amp;nbsp;</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

우리 연구소는 최근 자연 살해 (NK) 세포가 암 세포가 β - 갈 락토 - 결합 렉틴 galectin의 발현에 결함이 렌더링되는 경우 두개 내 생착 후 CNS-1 악성 신경 교종 곧 동소 이식 마우스 GL26과 쥐를 박멸 할 수 있음을 보여 주었다 -1 (GAL-1). 최근의 연구는 지금 GR-1 + /는 CD11b + 골수 세포의 인구는이 효과에 중요한 것을 알 수있다. 더 NK와 골수 세포가 우리가 단리 교종 함침 말초 혈 단핵 세포 (PBMC)의 분석을위한 광범위한 프로토콜을 개발했다 GAL-1 결핍 종양 거부를 부여하기 위해 협력하는 메커니즘을 이해하기. 이 방법의 종양 미세 환경 내로 침투 PBMC를 비교하여 여기에서 설명된다 shRNA를 녹다운 통해 GAL-1 결핍 렌더링하는 것과 GL26 교종을 GAL-1 발현. 프로토콜에 대한 설명으로 시작하는 방법 문화와 GL26 가전을 준비동계 C57BL / 6J 마우스의 뇌에 접종에 대한 재편. 그러므로 초기 뇌 종양 미세 환경에서 신경 교종 함침 된 PBMC의 분리 및 유세포 분석에 관련된 단계를 설명한다. 상기 방법은 뇌에 면역 침윤 시간에 데이터를 필요로하는 생체 내 실험 디자인에 적용 할 수있다. 교종 함침 된 PBMC는 독립적 인 실험 내내 시점 유사한 종양에서 관찰 유사한 세포 수와 24 시간 후 종양 생착 자마자 두개 내 종양으로부터 분리 될 수있어서, 민감하고 재현성이다. 실험자들은 단일 분석 될 샘플의 수에 따라 약 4-6 시간에 신경 교종 함침 된 PBMC의 세포 계측 분석을 흐르게 뇌 적출에서 방법을 수행 할 수있다. 대안 교종 모델 및 / 또는 셀 특정 검출 항체는 또한 몇몇 다른 immun의 침윤을 평가 experimentalists &#39;판단에 사용될 수있다전체적인 절차 변경 없이도 관심 E 세포 유형.

다음 게이팅 전략은 일반적인 실험에 사용되는 : FSC-SSC-대 → SSC-H 대 SSC-W → FSC-H FSC-W → CD45 대 대 카운트 → GR-1 대 CD11b를 → 카운트 대 NK1.1. 게이트는 GzmB 표현 (도 5a)에 기초하여 층화 GR-1 + / + CD11b를 골수 NK1.1 + 세포 및 NK 세포에 배치. NK1.1 + NK 세포 및 GR-1 + / CD11b를 SSC-A는 NK 세포의 작은 림프 크기 및 골수 세포의 비교적 큰 크기를 확인 대 FSC...

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

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.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

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

15.7K 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. 

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Percoll 그라디언트를 사용하여 두뇌와 척수 Mononuclear 세포의 분리

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

연구

면역학 및 감염병학

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

카르복시 Fluorescein Succinimidyl 에스터 (CFSE)를 사용 인간 Glioblastoma의 속도가 느린 - 나누면 전지의 식별 및 분리

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

의학

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

정렬 줄기 세포 Assays 및 흐름에 대한 기본 뇌 종양 조직의 처리

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the ...

암 연구학

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.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

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

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

유동 세포 계측법에 의한 중추 신경계에서 유래 된 대장 세포 및 단구 세포 유래 대 식세포의 분석

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

환자 Glioblastoma 절제술에서 세포 미세 환경의 정량적 면역 조직 화학

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

<meta charset="utf8"></head><body>Hyaluronan-Collagen I 하이드로겔 시스템으로 교모세포종 침습 복제

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

<meta charset="utf8"></head><body>저자 스포트라이트: 간질액 흐름을 통한 교모세포종 침습을 연구하기 위한 환자 정보 3D 모델

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

간질 흐름이 있는 침습적 신경교종 미세환경의 광중합 가능한 히알루론산-콜라겐 모델

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

Percoll 그라디언트를 사용하여 두뇌와 척수 Mononuclear 세포의 분리

카르복시 Fluorescein Succinimidyl 에스터 (CFSE)를 사용 인간 Glioblastoma의 속도가 느린 - 나누면 전지의 식별 및 분리

정렬 줄기 세포 Assays 및 흐름에 대한 기본 뇌 종양 조직의 처리

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

유동 세포 계측법에 의한 중추 신경계에서 유래 된 대장 세포 및 단구 세포 유래 대 식세포의 분석

환자 Glioblastoma 절제술에서 세포 미세 환경의 정량적 면역 조직 화학

실험적인 자가면역 뇌척수염 중 중추신경계에서 림프구 침투의 흐름 세포 분석

간질 흐름이 있는 침습적 신경교종 미세환경의 광중합 가능한 히알루론산-콜라겐 모델

간질 흐름이 있는 침습적 신경교종 미세환경의 광중합 가능한 히알루론산-콜라겐 모델