Name: obtaining human microglia from adult human brain tissue
Uploaded: 2020-08-30T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

SJ의 실험실은 IITJ의 기관 보조금으로 설립되었으며 생명공학부(BT/PR12831/MED/30/1489/2015)와 인도 전자정보기술부(No.4(16호)/2019-ITEA의 보조금으로 지원받고 있습니다. 인간의 뇌 조직 섹션은 기관 윤리위원회 허가 후 의학의 모든 인도 연구소 (AIIMS) Jodhpur에서 얻은. 비디오 그래피 지원을 위해 디자인 및 예술 협회 IIT Jodhpur의 B.Tech 학생 회원인 마야크 라토르에게 감사드립니다.

모든 조직은 인도 공과 대학 조드푸르와 모든 인도 의학 연구소 (AIIMS) Jodhpur의 연구소 윤리위원회에서 윤리적 허가 후 인수되었다.
1.조직 취득 및 처리(0일차)
<ol><li>얼음차가운 인공 뇌척수액(aCSF) 10mL(aCSF) (2mM CaCl2∙2H2O,10mM포도당, 3mM KCl, 26m MM NaHCO3,2.5mM NaHCO 3, 2.5mM NaHCO 3, 2.5mM MM MgCl2+6H42,202m)를 함유한50mL튜브?...

<ol>
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J., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+TBI+brain:+The+good,+the+bad,+and+the+dysregulated.">Microglia in the TBI brain: The good, the bad, and the dysregulated.</a> Experimental Neurology. 275 (03), 316-327 (2016).</li><li>Inoue, K., Tsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+neuropathic+pain:+cellular+and+molecular+mechanisms+and+therapeutic+potential.">Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential.</a> Nature Reviews Neuroscience. 19 (3), 138-152 (2018).</li><li>Bellver-Landete, V., 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=Microglia+are+an+essential+component+of+the+neuroprotective+scar+that+forms+after+spinal+cord+injury.">Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.</a> Nature Communications. 10 (1), 518 (2019).</li><li>Gutmann, D. H., 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/Brain+Macrophages+as+Central+Drivers+of+Brain+Tumor+Pathobiology.">Microglia/Brain Macrophages as Central Drivers of Brain Tumor Pathobiology.</a> Neuron. 104 (3), 442-449 (2019).</li><li>Smith, A. M., Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+side+of+microglia.">The human side of microglia.</a> Trends in Neurosciences. 37 (3), 125-135 (2014).</li><li>Galatro, T. 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nature Neuroscience. 20 (8), 1162-1171 (2017).</li><li>Friedman, B. 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=Diverse+Brain+Myeloid+Expression+Profiles+Reveal+Distinct+Microglial+Activation+States+and+Aspects+of+Alzheimer's+Disease+Not+Evident+in+Mouse+Models.">Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models.</a> Cell Reports. 22 (3), 832-847 (2018).</li><li>Rustenhoven, 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=PU.1+regulates+Alzheimer's+disease-associated+genes+in+primary+human+microglia.">PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.</a> Molecular Neurodegeneration. 13 (1), 44 (2018).</li><li>Sierra, A., Gottfried-Blackmore, A. C., McEwen, B. S., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55 (4), 412-424 (2007).</li><li>Mizee, M. 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=Isolation+of+primary+microglia+from+the+human+post-mortem+brain:+effects+of+ante-+and+post-mortem+variables.">Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables.</a> Acta Neuropathologica Communications. 5 (1), 16 (2017).</li><li>Rustenhoven, 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=Isolation+of+highly+enriched+primary+human+microglia+for+functional+studies.">Isolation of highly enriched primary human microglia for functional studies.</a> Scientific Reports. 6 (1), 19371 (2016).</li><li>Spaethling, J. 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=Primary+Cell+Culture+of+Live+Neurosurgically+Resected+Aged+Adult+Human+Brain+Cells+and+Single+Cell+Transcriptomics.">Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.</a> Cell Reports. 18 (3), 791-803 (2017).</li><li>Olah, 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=An+optimized+protocol+for+the+acute+isolation+of+human+microglia+from+autopsy+brain+samples.">An optimized protocol for the acute isolation of human microglia from autopsy brain samples.</a> Glia. 60 (1), 96-111 (2012).</li><li>Jha, 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=The+Inflammasome+Sensor,+NLRP3,+Regulates+CNS+Inflammation+and+Demyelination+via+Caspase-1+and+Interleukin-18.">The Inflammasome Sensor, NLRP3, Regulates CNS Inflammation and Demyelination via Caspase-1 and Interleukin-18.</a> The Journal of Neuroscience. 30 (47), 15811 (2010).</li><li>Freeman, 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=NLR+members+NLRC4+and+NLRP3+mediate+sterile+inflammasome+activation+in+microglia+and+astrocytes.">NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.</a> Journal of Experimental Medicine. 214 (5), 1351-1370 (2017).</li><li>Plant, S. 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=Lymphotoxin+beta+receptor+(Lt+betaR):+dual+roles+in+demyelination+and+remyelination+and+successful+therapeutic+intervention+using+Lt+betaR-Ig+protein.">Lymphotoxin beta receptor (Lt betaR): dual roles in demyelination and remyelination and successful therapeutic intervention using Lt betaR-Ig protein.</a> The Journal of Neuroscience. 27 (28), 7429-7437 (2007).</li><li>Arnett, H. 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=The+Protective+Role+of+Nitric+Oxide+in+a+Neurotoxicant-+Induced+Demyelinating+Model.">The Protective Role of Nitric Oxide in a Neurotoxicant- Induced Demyelinating Model.</a> The Journal of Immunology. 168 (1), 427 (2002).</li><li>Arnett, H. 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=TNF&#945;+promotes+proliferation+of+oligodendrocyte+progenitors+and+remyelination.">TNF&#945; promotes proliferation of oligodendrocyte progenitors and remyelination.</a> Nature Neuroscience. 4 (11), 1116-1122 (2001).</li><li>Trouplin, V., 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=Bone+marrow-derived+macrophage+production.">Bone marrow-derived macrophage production.</a> Journal of Visualized Experiments. (81), e50966 (2013).</li><li>Boltz-Nitulescu, G., 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=Differentiation+of+Rat+Bone+Marrow+Cells+Into+Macrophages+Under+the+Influence+of+Mouse+L929+Cell+Supernatant.">Differentiation of Rat Bone Marrow Cells Into Macrophages Under the Influence of Mouse L929 Cell Supernatant.</a> Journal of Leukocyte Biology. 41 (1), 83-91 (1987).</li><li>Englen, M. D., Valdez, Y. E., Lehnert, N. M., Lehnert, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte/macrophage+colony-stimulating+factor+is+expressed+and+secreted+in+cultures+of+murine+L929+cells.">Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells.</a> Journal of Immunological Methods. 184 (2), 281-283 (1995).</li></ol>

Microglia는 정상적인 뇌의 항상성을 보장하고 다양한 신경 질환4의 병리4생리학에서 중심적인 역할을한다. Microglia는 신경 발달과 시냅스2의형성의 중심입니다. Microglial 연구는 다양한 신경질환4의발달과 진행을 이해하는 데 중추적 인 것으로 입증되었습니다. 설치류 microglia는 1 차적인 microglial 연구 결과를 위한 선택의 널리 퍼진 모형입니다, ?...

중추 신경계(CNS)는 뉴런 과 신경교 세포1의복잡한 네트워크로 구성된다. 신경교 세포 중, 마이크로글리아는 CNS2,,3의타고난 면역 세포로서 기능한다. Microglia는 건강한 CNS4에서항상성을 유지하는 데 책임이 있습니다. Microglia는 또한 시냅스2를가지치기하여 신경 발달에 중요한 역할을합니다. Microglia는 여러 신경 질환의 병리생리학의 중심이지만 제한되지 는 않습니다. 알츠하이머병5,파킨슨병6,뇌졸중7,다발성 경화증8,외상성 뇌손상9,신경병성 통증10,척수 손상11, 진모(12) 등의 뇌종양이 있다.12
CNS 항상성 및 질병과 관련된 연구는 비용 효율적이고 시간 효율적인 인간 1 차 적인 microglia 격리 프로토콜13의땅때문에 설치류 microglia를 이용합니다. 설치류 마이크로글리아는 Iba-1, PU.1, DAP12 및 M-CSF 수용체와 같은 유전자의 발현에서 1차 인간 마이크로글리아를 유사하게 하며, 병든뇌(13)뿐만아니라 정상을 이해하는 데 효과적이었다. 흥미롭게도, TLR4, MHC II, 시글리크-11 및 시그렉-3과 같은 여러 면역 관련 유전자의 발현은 인간과 설치류마이크로글리아(13)사이에 다릅니다. 여러 유전자의 발현은 또한 두 종14,,15에서측현현 및 신경 퇴행성 질환에서 변화한다. 이러한 중요한 차이는 인간 microglia 항상성 및 질병에 있는 microglia 기능을 공부하는 필수적인 모형을 만듭니다. 1차 인간 마이크로글리아는 잠재적약물후보(16)의전임상 검진을 위한 효과적인 도구가 될 수 있다. 위에서 언급 한 이유는 기본 인간 microglia의 격리를위한 비용 효율적인 프로토콜에 대한 필요성을 강조한다.
우리는 종양 절제술 또는 그밖 외과 절제술을 위해 생성된 외과 창결과로 집합된 성인 인간 두뇌 조직에서 1 차적인 인간 microglia의 격리를 위한 프로토콜을 개발했습니다. 여기서 방법은 기존 방법과 상당히 다릅니다. 우리는 조직 수집 현장에서 실험실에서 격리 프로토콜을 시작하는 약 75 분의 전송 시간 후에 격리하고 배양 microglia를 할 수 있었습니다. 우리는 격리 된 microglia의 성장을 촉진하기 위해 L929 섬유 아세포 세포의 상체를 사용했습니다. 이 방법은 특히 1 차 microglia의 문화와 개발에 초점을 맞추고 있습니다. 준비된 결과 배양은 약 80% 마이크로글리아입니다. 다른 프로토콜은 밀도 그라데이션 원심 분리, 유동 세포측정 및 자기 구슬에 의해 미세글리아의 농축 된 배양을 제공하지만, 프로토콜은 1차,인간 microglia17,18,19,,20을배양하는 신속하고 간단하며 견고하며 비용 효율적인 방법입니다., 시체에서 고정 된 뇌 조직 대신 외과적으로 제거 된 살아있는 성인 뇌 조직을 활용하는 능력은 기존 절차(18,,21)와는 대조적으로이 방법의 추가 이점을 입증한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Himedia</td>
			<td>A002</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (4 &#176;C)</td>
			<td>Sigma</td>
			<td>146532</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Abdos</td>
			<td>P10203</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>New Brunswik</td>
			<td>Galaxy 170 S</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Himedia</td>
			<td>GRM077</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium with glutamine</td>
			<td>Himedia</td>
			<td>AL007S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Himedia</td>
			<td>RM9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Flacon tube (50 ml)</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>50CD1058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Ricinus communis agglutinin-1</td>
			<td>Vector</td>
			<td>FL-1081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DM2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>GA5</td>
			<td>3670S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswik Scientific</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC&#160;CCL-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar air flow</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Himedia</td>
			<td>MB040</td>
			<td></td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>Merck</td>
			<td>567545</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Mixture F-12 Ham Medium</td>
			<td>Himedia</td>
			<td>Al106S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Duran Group</td>
			<td>237554805</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Himedia</td>
			<td>ML023</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Himedia</td>
			<td>MB043</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Labware</td>
			<td>LW-SP1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Himedia</td>
			<td>MB045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Himedia</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.2&#956;, 25 mm diameter)</td>
			<td>Axiva</td>
			<td>SFPV25R</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 tissue culture flasks suitable for adherent cell culture.</td>
			<td>Himedia</td>
			<td>TCG4-20X10NO</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco&#160;</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining human microglia from adult human brain tissue

Microglia는 중추 신경계 (CNS)의 거주자 타고난 면역 세포입니다. Microglia는 개발 중, 항상성을 유지 하며 감염 또는 부상 중에 중요한 역할을 합니다. 몇몇 독립적인 연구 단은 microglia가 자기 면역 질병, 자가 염증성 증후군 및 암에서 재생하는 중앙 역할을 강조했습니다. 몇몇 신경학상 질병에 있는 microglia의 활성화는 병원성 프로세스에 직접 참여할 수 있습니다. 1 차적인 microglia는 두뇌에 있는 면역 반응을 이해하는 강력한 공구입니다, 세포 세포 상호 작용 및 질병에 있는 dysregulated microglia 표현형. 1 차적인 microglia는 불멸한 microglial 세포주 보다는 더 나은 생체 내 microglial 속성에서 모방합니다. 인간 성인 microglia 인간의 태아와 설치류 microglia에 비해 뚜렷한 속성을 전시. 이 프로토콜은 성인 인간의 뇌에서 기본 마이크로 글리아의 격리를위한 효율적인 방법을 제공합니다. 이러한 microglia를 연구 하는 microglia와 CNS에 있는 다른 상주 세포 인구 사이 세포 세포 상호 작용에 중요 한 통찰력을 제공할 수 있습니다., oligodendrocytes, 뉴런 및 성상 세포. 또한, 다른 인간의 두뇌에서 microglia 개인화 된 약및 치료 응용 프로그램의 무수한에 대 한 독특한 면역 반응의 특성화를 위해 배양 될 수 있습니다.

위에서 언급한 프로토콜(도1)을사용하여, 우리는 1차 인간 마이크로글리아를 외과적으로 절제된 뇌 조직으로부터 분리할 수 있었다. 배양된 세포는 미세글리아(green)에 대한 리시누스 공동체 agglutin-1(RCA-1) 렉틴과 성상세포(red)(도 2)를 위해 염색하였다(도2)이전에 설명된 바와 같이22,,<sup class="xref...

Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

Obtaining Human Microglia from Adult Human Brain Tissue

이 프로토콜은 살아있는, 성인, 인간의 뇌 조직에서 기본 microglia를 격리하는 효율적이고 비용 효율적이며 강력한 방법입니다. 고립 된 1 차적인 인간 microglia는 항상성 및 질병에 있는 세포 프로세스를 공부하기 위한 공구역할을 할 수 있습니다.

성인 인간 뇌 조직에서 인간 마이크로글리아 획득

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

프로토콜의 전반적인 목표는 살아있는 성인 인간 두뇌 조직에서 1 차적인 microglia 세포를 격리하는 것입니다. 우리의 경우, 이것은 수술 중 수술 창으로 수집되었습니다. 이것은 처음에 얼음 차가운 PBS에 있는 두뇌 조직을 수집해서 달성됩니다, 조직은 그 때 1 mm 큐브 작은 조각으로 다진되고, 난 그냥 트립신 EDTA의 도움으로 했습니다.이에 따라, 세포는 원심분리에 의해 수확되고 48시간 동안 안드로겐 세포 배양에 적합한 플라스크로 도금된다. 세포는 플라스크에서 다시 한 번 수확되고 추가 사용까지 배양됩니다. 1차 인간 마이크로글리아 세포는 이제 면역 세포 화학을 사용하여 추가 실험을 위해 사용될 수 있다.성인 인간의 뇌 조직에서 microglia 세포를 격리하기위한 우리의 프로토콜의 주요 장점은 효과적으로 매우 비용 효율적인 방법으로 기본 성인 인간 microglia 세포를 제공한다는 것입니다. 프로토콜은 견고하며 기존 프로토콜에서 사용되는 단계 수를 줄여 셀 손실을 줄입니다. 얼음 차가운 aCSF의 10 mL를 포함하는 50 mL 팔콘 튜브에서 조직을 수집합니다.수집 튜브를 70% 알코올로 조심스럽게 닦고 무균 라미나르 공기 흐름 챔버로 옮겨보세요. ACSF를 신중하게 폐기하고 팔콘 튜브에서 조직을 계량합니다. 빈 팔콘 튜브의 무게를 추론하여 조직의 약 중량을 계산합니다.따뜻한 신선한 aCSF에서 섭씨 37도까지. 5 분 동안 따뜻한 aCSF에 조직을 유지합니다. 이 단계는 세포 사멸을 피하기 위해 중요합니다.aCSF를 신중하게 폐기하십시오. 그리고 1X PBS로 한 번 티슈를 37도까지 따뜻하게 씻어 낸다. 필요에 따라 반복되는 PBS 세척으로 모든 혈액을 씻어 내도록 하십시오.5 분 동안 따뜻한 PBS에 조직을 배양. PBS의 절반을 신중하게 폐기하십시오. 남은 PBS와 함께 멸균된 페트리 접시에 티슈를 옮길 수 있습니다.1mL 파이펫으로 남은 PBS를 조심스럽게 제거합니다. 이것은 조직의 손실을 방지 할 것이다. 멸균 메스를 사용하여 적어도 1mm 큐브 작은 조각으로 발행 주사위.페트리 접시에 0.25%의 트립신 EDTA 2mL을 넣고 파이펫의 도움으로 접시를 철저히 씻으십시오. 다진 조직을 그램 조직당 10mL를 함유하는 50mL 팔콘 튜브로 0.25%의 트립신 EDTA로 옮김한다. 10mL 세로지오피펫을 통해 파이펫팅하여 섞는다.37도 섭씨와 250 rpm의 속도로 30 분 동안 셰이커에 튜브를 배양하십시오. 트립신을 중화하기 위해 중간 중화 10mL를 추가합니다. 10mL 세로지오피펫과 철저히 섞으세요.10 분 동안 섭씨 4도에서 2000G에서 튜브를 원심 분리합니다. 상체를 버리고 배양 배지의 1 mL에서 펠릿을 다시 중단하십시오. 안드로겐 세포에 적합한 T25 플라스크에 세포를 놓고 4mL의 추가 배양 배지를 추가한다.배양 배지는 20%L929 상류제 와 1%연쇄절제술을 함유할 것이다. 주식 문화 배지에 추가하는 대신 플라스크에 L929 상체를 별도로 추가하는 것이 좋습니다. 우리는 균질적으로 조직을 처분하는 플라스크로 완성됩니다.오염의 가능성을 증가시킬 수 있기 때문에 흔들리는 동안 플라스크의 목에 미디어를 가져 오지 마십시오. 플라스크를 섭씨 37도에서 48시간 동안 5%CO2로 배양합니다. 원심분리기 튜브에서 0일째에 준비된 T25 문화 플라스크에서 미디어를 수집합니다.수집된 미디어를 4도 에서 4분 동안 섭씨 4도에서 원심분리합니다. 수집된 매체가 원심 분리되는 동안, 1X PBS로 1회 0플라스크를 세척하십시오. 플라스크를 부드럽게 흔들어 남은 조직 조각을 제거합니다.잔재된 조각이 문화에 부정적인 영향을 미치지 않기 때문에 플라스크의 가혹한 흔들림을 피하십시오. 플라스크에 신선한 문화 매체 5mL를 추가합니다. 원심 분리 된 튜브에서 상체를 폐기하십시오.튜브 중 하나에 배양 매체 1mL을 추가하고 파이펫과 완전히 섞습니다. 연재하여 다른 튜브에 셀이 혼합 된 미디어를 추가하고 완전히 섞습니다. 안드로겐 세포에 적합한 별도의 T25 플라스크에 세포를 놓습니다.플라스크에 신선한 문화 매체 4mL를 추가합니다. 플라스크를 섭씨 37도에서 48시간 동안 5%CO2로 배양합니다. 플라스크에서 미디어를 폐기하고 신선한 5mL 문화 매체를 추가합니다.플라스크를 섭씨 37도에서 48시간 동안 5%CO2에서 배양합니다. 6 일째에, 세포는 추가 실험을 위한 준비가 될 것입니다. 여기에 표시된 이미지에서.섹션의 첫 번째 패널 GFAP로 염색 된 성상 세포, 녹색 색상. 두 번째 패널에서, 섹션 RCA와 마이크로 글리아 얼룩의, 녹색 색상. 섹션 B 마이크로글리아에서 RCA로 염색, 이는 색상에 녹색, 그리고 성상 세포는 GFAP로 염색, 색상에 빨간색.이미지의 오버레이는 마이크로글리아와 성상세포가 함께 표시됩니다. 준비된 배양에 있는 세포의 대다수가 microglia이다는 것은 심상에서 분명합니다. 이미지는 시각 장애인 제어에 의해 정량화되었고 그 결과는 C.섹션C.에서 표현되는 결과는 제조된 배양에서 세포의 약 80%가 마이크로글리아 세포임을 보여주었다.이 기술은 약 1 시간 반에서 수행 할 수 있습니다. 이 절차에 따라 나는 다운 스트림 실험에 더 사용할 수있는 성인 인간의 뇌 조직에서 priming microglia를 선택하는 방법의 좋은 이해가 있어야합니다.

obtaining-human-microglia-from-adult-human-brain-tissue

Research

JoVE Journal

Neuroscience

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

결합 컴퓨터 게임 기반 고밀도 뇌파와 적외선 가즈 추적 Behavioural 실험

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

연구

신경과학

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

뇌 조직의 만성 Optogenetic의 자극을위한 광섬유 주입

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

신선한 인간의 뇌 조직에서 뇌 모세 혈관의 절연

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

절연 및 애벌레 Zebrafish 두뇌에서 신경 세포, 대 식 세포 및 Microglia RNA 추출

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

성인 인간의 뇌에서 단기 자유 부동 슬라이스 문화

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

깊은 단세포 RNA 염기서열 분석에 대한 하나의 성인 마우스 뇌 반구에서 영역 별 Microglia의 격리

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

형광 활성화 핵 분류를 사용하여 신선 냉동 피질로부터 성인 인간 성상세포 집단의 분리

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

인간 미세아교세포 유사 세포: 유도만능줄기세포로부터의 분화 및 인간 시냅토좀을 이용한 체외 생세포 식균작용 분석

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...