제복 차림의 서비스 대학 교내 프로그램에 의해 자금 지원.

1. 신생아 쥐 뇌 조직의 해부 <ol><li> 칠이 Leibovitz의 L-15 에어컨 미디어 (레이 보위 츠 L-15 + 0.1 % BSA + 1 %의 펜 / Strep) ~ 4 ° C. 따뜻한 문화 미디어 (DMEM + 10% FBS + 1% 페니실린 / 스트렙토 마이신) 37 가기 ° C. 얼음 냉방 L-15 미디어의 4-5 ML 4 개의 60x15 mm 배양 접시를 준비합니다. 100 % 에탄올 모든 수술 도구를 소독. </li><li> 70 % 에탄올과 P2 신생아 랩 새끼를 씻어. (P1-P5 사이에 세 신생아 쥐 새끼는이 프로토콜에서 사용할 수 있습니다.) </li><li> 신속 멸균 날카로운 가위로 강아지 쥐의 목을 벨 및 70 % 에탄올로 바로 머리를 놓습니다. 5 쥐 새끼의 총 반복 후 생리 식염수로 머리를 전송합니다. </li><li> 얼음 5 ML L-15 용액 (레이 보위 츠 L-15 + 0.1 % BSA + 1퍼센트 펜 / Strep) - 4 페트리 접시에 머리와 장소에서 전체 두뇌를 제거합니다. </li><li> 1.4 쓰레기의 나머지 새끼 용 - 절차 1.2 반복합니다. </li><li> 두뇌에서 meninges를 제​​거하고 DES를 전송L-15 솔루션의 4-5의 ML과 새로운 배양 접시에 ired 뇌 조직 (즉, 피질, 소뇌, 전체 뇌 등) (레이 보위 츠 L-15 + 0.1 % BSA + 1 %의 펜 / Strep) 얼음. </li></ol> 2. 혼합 Glial 세포 인구의 작성 <ol><li> 가위 혹은 칼날로 손상 뇌 세포없이는 무균 50 ML 원뿔 튜브에 10 ML의 피펫과 조직을 전송합니다. </li><li> 4 ° C.에서 5 분 2,500 RCF에 원뿔 원심 분리기 </li><li> 뜨는을 기음. 신선한 L-15 미디어의 4-5 ML 추가하십시오 (레이 보위 츠 L-15 + 0.1 % BSA + 1 %의 펜 / Strep). </li><li> 멸균 10 ML의 피펫과 피펫은 조직을 위아래로 10 회. </li><li> 신선한 50 ML 원뿔 튜브에 세포 스트레이너 (100 μm의 제품 모공)를 놓습니다. 멸균 5 ML 피펫으로하고 셀 스트레이너에 빠질때 피펫과 피펫 한 조직 위아래은 원뿔 튜브에 세포 스트레이너를 통해 자료를 분배. </li><li> 신선한 L-15 미디어의 4-5 ML (와 세포 스트레이너를 씻어 레이 보위 츠 L-15 + 0.1 % 학사+ 1 %의 펜 / Strep). </li><li> 4에서 5 분 동안 2,500 RCF에서 무한 세포와​​ 원뿔 원심 ° C. </li></ol> 3. 혼합 Glial 세포 문화 도금 및 유지 관리 <ol><li> 각 쥐​​ 새끼 머리가 처리된 경우, 문화, 미디어의 12 ML 추가하여 한 멸균 T-75 플라스크를 준비 (를 DMEM + 10% FBS + 1퍼센트 페니실린 / 스트렙토 마이신) 각 플라스크에.에게 </li><li> pelleted 세포에서 뜨는을 기음과 세포 펠렛으로 문화 미디어의 5-6 ML을 추가합니다. 10 ML의 피펫으로 10 배 아래로 피펫합니다. </li><li> 각 T-75 플라스크에 세포 현탁액의 동등한 볼륨을 전송합니다. </li><li> 1~3주 총 37 ° C에서 5 % CO 2 배양기에서 flasks을 품어. </li><li> Flasks는 처음 5 일 동안 incubated, 그리고 다섯 번째 날에, 신선한 매체의 12 ML 각 플라스크에 문화 미디어를 교체하고 인큐베이터로 돌아가려한다. 그런 다음, 매 3 일 합류를 달성하기위한 새로운 매체의 12 ML 각 플라스크에 갖추어진 미디어를 대체합니다. 이것은 어떻게해야NE는 매우 신중하게 세포 부착 flasks의 바닥에 닿지 않고. </li></ol> 4. 차 Microglia의 분리 및 도금 <ol><li> 혼합 glial 문화가 완전히 합류 후에는 인큐베이터에서 flasks를 제거하고 환경 공기 가스 교환을 방지하기 parafilm있는 술병 뚜껑을 포괄합니다. </li><li> 37에서 1 시간 동안 100 rps (랩 동반자 SI-600, Jeio 테크)에서 flasks 흔들어 ° C. </li><li> 플라스크 표면에 astrocyte 레이어를 중단하지 않고 10 ML의 피펫을 가진 모든 flasks에서 미디어를 수집합니다. 50 ML 원뿔 튜브에 미디어를 넣습니다. 인큐베이터에 flasks 및 반품 flasks에 신선한 미디어를 추가합니다. </li><li> 4에서 5 분 동안 2,500 RCF에서 원뿔 튜브를 원심 ° C. </li><li> 모든 원뿔 튜브의 표면에 뜨는을 기음. 세포 펠렛은 고순도 microglia 세포입니다. microglia 도금 미디어 (DMEM + 10 % FES + 1% 페니실린 / 스트렙토 마이신) 1 ML에 Resuspend 쥐똥. </li><li> 사용 resuspended 매체 셀 밀도를 세어hemocytometer. </li><li> ML 밀도 당 2 X 10e5 세포를 달성하기 위해 microglia 도금 매체의 적절한 볼륨을 추가합니다. 실험 분석을 위해 적절한 플레이트. </li><li> microglia는 하룻밤 사이에 부착하도록 허용합니다. </li></ol> 5. 대표 결과 프로토콜은 고순도 기본 microglia 문화에 결과를 위에서 설명한. 로 대식 세포 / 세포 microglia 특정 마커 (Iba1)에 immunohistochemistry에 의해 결정, 도금 microglia 문화 정보&gt; 90% 순수 (그림 2)입니다. 또한 astrocyte, oligodendrocyte 및 신경 세포 세포 특정 마커에 대해 더럽히는 것은 최소한의 오염 (그림 2)를 보여줍니다. 혼합 glial 세포 문화를 맺은 후, microglia은 4 연속 시간까지 흔들어에 의해 격리 수 있습니다. 초기 microglia 절연은 약 2.2 X 106 세포 / ML 또는 10 flasks 및 예상 수확량 약 9,000,000 세포가 각각의 연속된 SHA와 감소를 얻을 것입니다KE. 이 방법에 의해 격리 Microglia는 phagocytosis 능력과 같은 질소 산화물 생산과 같은 기능과 독성의 연결 (그림 3) 13-14를 조사하기 위해 성공적으로 사용되었습니다. <img alt="그림 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> 그림 1. 혼합 glial 세포 배양 및 기본 microglia의 절연을 준비하기위한 프로토콜의 개요. <img alt="그림 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> 그림 2. 같은 astrocyte microglia (Iba-1, 빨강), (GFAP, 녹색), 신경 세포 (NeuN, 빨강)와 oligodendrocyte를 사용 immunohistochemical 분석에 의해 검증 고순도 microglia의 절연 (CC1, 빨강) 특정 마커. DAPI (파란색)에 의해 문화에 세포의 DNA를 염색법도 표시됩니다. <img alt="그림 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> 그림 3. Microglia는 방법 descri에 고립침대를 포함한 assays의 숫자에 사용하지만, phagocytosis의 assays, 함수 assays와 neurotoxicity 연구에 국한되지되었습니다. 이러한 연구에서 우리는 () 또는 LPS (B) 처리 미디어, 계량하실 수 있습니다 fluorescently 레이블 라텍스 구슬 (빨간색), (그들의 phagocytosis를 증가 microglia (Iba-1 양성, 녹색)를 통제할 수 있도록 노출된 경우 것으로 나타났습니다 C). 또한, LPS (1ng/ml) 치료는 microglia (D)에서 질소 산화물의 생산을 늘릴 수 있습니다. 마지막으로, transwell 삽입 구분 또는 직접 microglia - 신경 세포 배양 모두를 통해, 우리는 락트 산 탈수소 효소 (LDH) 릴리스 (E, F)에 의해 측정으로 LPS와 incubated microglia가의 연결을 세포 죽음을 유도 것으로 나타났습니다.

<ol>
	<li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu. Rev. Immunol. 27, 119-145 (2009).</li><li>Raivich, G., Bohatschek, M., Kloss, C. U., Werner, A., Jones, L. L., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroglial+activation+repertoire+in+the+injured+brain:+graded+response,+molecular+mechanisms+and+cues+to+physiological+function.">Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function.</a> Brain Res. Brain Res. Rev. 30, 77-105 (1999).</li><li>Loane, D. J., Byrnes, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microglia+in+neurotrauma.">Role of microglia in neurotrauma.</a> Neurotherapeutics. 7, 245-265 (2010).</li><li>Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+inflammation+in+neurodegeneration.">Mechanisms underlying inflammation in neurodegeneration.</a> Cell. 140, 918-934 (2010).</li><li>Pocock, J. M., Liddle, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+signalling+cascades+in+neurodegenerative+disease.">Microglial signalling cascades in neurodegenerative disease.</a> Prog. Brain Res. 132, 555-565 (2001).</li><li>Block, M. L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+microglial+activation+and+progressive+dopaminergic+neurotoxicity.">Chronic microglial activation and progressive dopaminergic neurotoxicity.</a> Biochem. Soc. Trans. 35, 1127-1132 (2007).</li><li>Barger, S. W., Basile, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+microglia+by+secreted+amyloid+percursor+protein+evokes+release+of+glutamate+by+cystine+exchange+and+attenuates+synpatic+function.">Activation of microglia by secreted amyloid percursor protein evokes release of glutamate by cystine exchange and attenuates synpatic function.</a> J. Neurochem. 76, 846-854 (2001).</li><li>Ni, M., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+microglia:+isolation,+culturing,+and+selected+applications.">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Curr. Protoc. Toxicol. Chapter 12, Unit 12.17 (2010).</li><li>Hassan, N. F., Rifat, S., Campbell, D. E., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+flow+cytometric+characterization+of+newborn+mouse+brain-derived+microglia+maintained+in+vitro.">Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.</a> J. Leukoc. Biol. 50, 86-92 (1991).</li><li>Hassan, N. F., Prakash, K., Chehimi, J., McCawley, L. J., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+newborn+rabbit+brain-derived+microglia.">Isolation and characterization of newborn rabbit brain-derived microglia.</a> Clin. Immunol. Immunopathol. 59, 426-435 (1991).</li><li>Hassan, N. F., Campbell, D. E., Rifat, S., Douglas, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+fetal+brain-derived+microglia+in+in+vitro+culture.">Isolation and characterization of human fetal brain-derived microglia in in vitro culture.</a> Neuroscience. 41, 149-158 (1991).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:+immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li><li>Byrnes, K. R., Stoica, B., Loane, D. J., Riccio, A., Davis, M. I., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptor+5+activation+inhibits+microglial+associated+inflammation+and+neurotoxicity.">Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity.</a> Glia. 57, 550-560 (2009).</li><li>Loane, D. J., Stoica, B. A., Pajoohesh-Ganji, A., Byrnes, K. R., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+metabotropic+glutamate+receptor+5+modulates+microglial+reactivity+and+neurotoxicity+by+inhibiting+NADPH+oxidase.">Activation of metabotropic glutamate receptor 5 modulates microglial reactivity and neurotoxicity by inhibiting NADPH oxidase.</a> J. Biol. Chem. 284, 15629-15639 (2009).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

이 프로토콜은 정기적으로 연구 실험을위한 순수하고 건강한 microglia를 생성하기 위해 이용되는 동안 절차의 중요한 부분 중에 기술적인 관점의 신중한 검토가 고립된 microglia의 변화를 최소화합니다. 첫째, 제시간에 작업 신생아 쥐 새끼의 뇌 조직의 해부 동안 조직에 hypoxic 및 허혈성 손상을 최소화해야합니다. 그러나 meninges의 존재는 이러한 문화 조건 하에서 그들의 빠른 확산 속도로 인해 상?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 60mm X 15mm 배양 접시 </td><td> 피셔 브랜드 </td><td> 0875713A </td><td></td></tr><tr><td> 샤프 해부 가위 </td><td> 파인 과학 도구 </td><td> 14094-11 </td><td></td></tr><tr><td> 뒤몽 # 곡선 7B 포셉 표준 팁, 11cm </td><td> 파인 과학 도구 </td><td> 11270-20 </td><td></td></tr><tr><td> 뒤몽 # 5 포셉 표준 팁, 스트레이트, 11cm </td><td> 파인 과학 도구 </td><td> 11251-10 </td><td></td></tr><tr><td> 50 ML 원추형 원심 분리기 튜브 </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ML serological pipettes </td><td> Grenier 바이오 하나 </td><td> 606,180 </td><td></td></tr><tr><td> 10 ML serological pipettes </td><td> Grenier 바이오 하나 </td><td> 607,180 </td><td></td></tr><tr><td> 100 μm의 멸균 나일론 셀 스트레이너 </td><td> 매 </td><td> 35-2360 </td><td></td></tr><tr><td> 75cm 두 조직 문화 flasks </td><td> 코닝 </td><td> 430,641 </td><td></td></tr><tr><td> Dulbecco의 최소한의 필수적인 매체 (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz의 L-15 배지 </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> 태아 말 혈청 </td><td> 어부 </td><td> SH3007402 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> 100 % 에탄올 </td><td> 워너 그레이엄 회사 </td><td> 64-17-5 </td><td></td></tr><tr><td> 인산염은 10X 생리 식염수, 산도 7.4 버퍼 </td><td> 품질 생물학, INC. </td><td> 119-069-131 </td><td> 멸균, 증류수에 1X </td></tr><tr><td> 바이오 해저드 가방 </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> 혈구계 </td><td> Hausser 과학 </td><td> 1492 </td><td></td></tr><tr><td> cellBIND 표면과 6도 세포 배양 접시 </td><td> 코닝 </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

포유류의 두뇌에서 전체 세포 인구의 약 12​​ %를 Microglia 계정. 뉴런과 astrocytes는 신경 계통의 주요 세포 유형을 고려하는 동안 microglia는 파편과 병원균을 위해 조직을 모니터링하고 phagocytic 활동 1,2를 통해 실질 조직의 항상성을 유지하여 정상적인 뇌 생리에 중요한 역할을한다. Microglia는 neurodegenerative 질병, 외상성 뇌 손상, 그리고 신경계 감염 3를 포함해 부상과 질병 조건의 수를 동안 활성화됩니다. 이러한 작동 조건에서 microglia은 그들의 phagocytic 활동을 증가 morpohological과 proliferative 변화를 받아야하고, 적극적으로 반응 산소와 질소 종, 프로 - 염증 chemokines 및 크린 시토킨을 분비, 종종 paracrine 또는 autocrine 루프 4-6를 가동합니다. 이 microglial 답변 신경학 조건 질병 pathogenesis에 기여으로 연구 m에 초점을icroglia이 보증됩니다. 두뇌의 세포 이질 때문에, 그것은 생체내 실험의 도중에 고순도로 충분한 microglial 샘플 자료를 얻기 위해 기술적으로 어렵습니다. microglia의 neuroprotective과 neurotoxic 기능에 관한 현재의 연구는 지속적으로 연구를위한 충분한 수율와 순수하고 건강한 microglia를 생성하는 루틴 기술적 방법을 필요로합니다. 우리는, 텍스트 및 비디오에 다운 스트림 애플 리케이션의 다양한 혼합 glia 문화에서 순수 일차 microglia를 분리하기위한 프로토콜을 제시한다. 간단히,이 기술은 혼합 glial 세포 문화를 만들어 신생아 쥐 새끼에서 dissociated 뇌 조직을 활용합니다. 혼합 glial 문화 confluency 도달 후, 일차 microglia는 기계 흔들림의 짧은 기간으로 문화에서 고립된다. microglia 그러면 실험적인 연구를위한 고순도로 도금된다. 이 방법론의 원리와 프로토콜되었습니다문헌 7,8에 설명되어. 또한, 기본 microglia를 분리하기 위해 대체 방법을 잘 9-12 설명되어 있습니다. 균질 뇌 조직은 기본 microglia 12 양보하는 밀도 기울기 원심 분리로 구분됩니다. 그러나, 원심 분리는 적당한 길이 (45 분)입니다 그리고 세포 손상 및 활성화뿐만 아니라, 풍부한 microglia 및 기타 세포 인구를 일으킬 원인이 될 수 있습니다. 다른 프로토콜은 문화 9-11에있는 동안 흔들림 (16 시간) 장기간에 의해 생물의 다양한 기본 microglia를 분리하기 위해 활용되었습니다. 떨고 후 미디어 뜨는는 microglia를 분리 centrifuged있다. 이것은 더 이상 두 단계의 격리 방법도 microglial 기능과 활성화를 교란시키다 있습니다. 우리는 주로 여러 가지 이유로 저희 연구실에서 다음과 microglia 격리 프로토콜을 활용 (1) 기본 microglia (2 더 충실 불후의 쥐 microglia 세포 라인보다 생체내 생물학에서 시뮬레이션) 공칭 기계적 장애는 잠재적인 세포 기능 장애 또는 활성화를 최소화하고, (3) 충분한 수율은 혼합 glial 세포 문화가 통과하지 않고도 얻을 수있다. 그것은이 프로토콜이 microglia를 분리하기 위해 신생아의 쥐 새끼의 뇌 조직을 사용하고 microglia 상당히 고립된 microglia의 이윤율, 정품 인증 상태와 기능적 특성에 영향을 미칠 수있는 분리 이전 쥐를 사용한다는 점에 유의하는 것이 중요하다. 노화가 microglia 부전, 증가 neuroinflammation 및 neurodegenerative pathologies와 연결되어있다는 증거가있다, 그래서 이전의 연구는 노화가 중요한 매개 변수는 어디에 좋은 neurodegenerative 질병에서 microglia의 역할을 이해하기 위해 전직 생체내 성인 microglia를 사용했습니다. 그러나 전직 생체내 microglia는 시간의 장시간 기간 동안 문화에 보관하실 수 없습니다. 이 프로토콜은 문화의 기본 microglia의 수명을 연장하면서 따라서, 그것은 microglia가 D를 행동 것을인지해야생체내 설정에서로 번역하면 ifferently 성인 microglia에서와 체외 연구에서 신중하게 고려되어야합니다.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

두뇌의 세포 이질에서 기본 microglia를 분리하는 것은 생리적 및 병리 학적 조건에 모두 자신의 역할을 조사하는 것이 필수적이다. 이 프로토콜은 기계적인 고립과 높은 수율과 높은 순도를 제공 혼합 세포 배양 기술에 대한 실용적 기본 microglial 세포에 대해 설명<em&gt; 체외에서</em&gt; 학업 및 다운 스트림 응용 프로그램입니다.

신생아 쥐 뇌 조직의 혼합 Glial 세포 배양의 기본 Microglia 격리

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K 조회수.  Uniformed Services University. 두뇌의 세포 이질에서 기본 microglia를 분리하는 것은 생리적 및 병리 학적 조건에 모두 자신의 역할을 조사하는 것이 필수적이다. 이 프로토콜은 기계적인 고립과 높은 수율과 높은 순도를 제공 혼합 세포 배양 기술에 대한 실용적 기본 microglial 세포에 대해 설명 체외에서 학업 및 다운 스트림 응용 프로그램입니다.

비디오: Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

성인 간세포의 혼합 차 문화에서 Macrophages을 분리하는 간단하고 효율적인 방법

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

연구

면역학 및 감염병학

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

기본 인적 Keratinocytes에서 Organotypic 뗏목 문화의 생성

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

신생아와 성인 중추 신경계에서 배양 소교 세포

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

쥐과 신생아에서 보존 면역 표현형과 기능으로 두피 소교 세포의 분리

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

쥐 고환의 세르 톨리 (Sertoli) 세포의 분리 및 세뇨관 세포

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

마우스에서 차 간세포 문화에 염증 반응의 유도

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

처음 초 유 수 유 전후 개별 Oxytocin 분 비 뇌 핵 신생아 쥐에 있는 염증과 세포 스트레스 평가

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

원생 동물 기생충 감염에 대 한 기본 성상 세포와 Microglia의 수반되는 격리

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...