Name: analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry
Uploaded: 2017-06-22T14:00:00.000+00:00
Duration: 10 min 43 s
Description: Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

이 작품은 Agence Nationale Pour la Recherche (ANR-12-MALZ-0003-02-P2X7RAD), 협회 France Alzheimer 및 Bpifrance의 교부금으로 지원되었습니다. 우리 연구소는 Inserm, CNRS, Université Pierre et Marie-Curie 및 &quot;Investissements d&#39; avenir&quot;ANR-10-IAIHU-06 (IHU-A-ICM) 프로그램도 지원합니다. CELIS 세포 배양 핵심 시설의 도움에 감사드립니다.

여기에 설명 된 모든 방법은 ICM 연구소의 기관 동물 관리 및 사용위원회와 다윈 프랑스 동물 동물위원회의 승인을 받았으며 프로토콜 01407.02로 보호됩니다. 1. 준비 <ol><li> 각 마우스에 다음을 결합하여 1.5 ML 튜브에서 소화 칵테일을 준비하십시오 : 1 ML 인산염 완충 식염수 (PBS); 13 wunsch / mL (원액), 최종 농도 1.6 wunsch / mL에서 123 μL 소화 효소 (물질 표 참조); 및 100 mg / mL (원액), 최종 농도 0.5 mg / mL에서 5 μL DNase I (물질 표 참조). </li></ol> 2. 관류 및 해부 <ol><li> 치사량의 pentobarbital (400 μg / mL에서 100 μL)로 마우스를 안락사하십시오. </li><li> 동물을 앙와위에 놓고 70 % 에탄올로 몸을 적시십시오. </li><li> xyphoid 프로세스 바로 아래의 미세 가위로 복부 피부를 잘라 흉부 캐비닛을 노출시킵니다.와이. </li><li> 횡격막을 절개하고 가슴을 노출시키기 위해 주둥이 방향에서 꼬리 방향으로 미세한 가위로 늑골의 측면을 잘라냅니다 (다른 장기 절단을 피하십시오). 좌심실 (LV)과 우심방 (RA)을 확인하십시오. </li><li> LV 내부에 25G 바늘이 달린 나비 카테터를 삽입하십시오. 미세 가위로 RA를 절개하고 혈류가 시작될 때 혈관에서 세포와 혈액을 제거하기 위해 20 mL PBS로 LV를 통해 10 mL / 분 유속으로 관류를 시작하십시오. 성공적인 관류는 혈액이 옮겨지면서 폐와 간이 희석되어 나타납니다. </li><li> 중간 가위로 동물의 목을 베십시오. 척추의 해부학적인 측면에서 복부 근육 조직을 절제하고 꼬리의 기저부에서 척추를 절단하여 척추를 정밀한 가위로 해부하여 체내에서 분리합니다. </li><li> 요추 측면에 PBS와 마운트 200 μL 피펫 팁을 포함하는 10 ML 주사기를 삽입척수를 제거하고 척수를 자궁 경부에 플러시하십시오. 참고 : 200 μL 피펫 팁은 가장 넓은 끝 (약 1 cm)에서 가위로 자르고 이전에 PBS로 채워진 10 mL 주사기에 단단히 삽입됩니다. </li><li> 두개골 위의 피부를 제거하고 포셉을 사용하여 뇌를 노출시킵니다. 두개골 바닥에 가위 날을 넣고 화살 봉합 후 두개골을 자릅니다. 작은 포셉을 절단 부위에 넣고 부드럽게 두개골을 들어 올리십시오. 마우스 머리에서 두뇌를 해부하고 후각 구근과 소뇌를 제거합니다. </li><li> 소화 칵테일 (단계 1.1에서) 1.128 ML PBS를 포함 1.5 ML 튜브에 뇌와 척수를 수집합니다. </li></ol> 3. 세포 해리 <ol><li> 작은 가위 ( 그림 1 )로 1-2 밀리미터 3 조각으로 1.5 ML 튜브 (단계 2.9)에서 뇌와 척수를 미세하게 자르십시오. </li><li> 인큐베이터에서 30 분 동안 다진 조직이 들어있는 1.5 ML 튜브를 품어5 % CO2로 37 ° C에서 건조시킨다. 참고 :이 단계에서 밀도 그래디언트를 준비하십시오. </li><li> 효소 반응을 중지 1.5 ML 튜브에 20 μL 0.5 M EDTA (재고 솔루션), 최종 농도 10 MM을 추가합니다. </li><li> 부드럽게 5 ML 피펫으로 위아래로 pipetting하여 세포 현탁액을 확인하십시오. </li><li> 10 ML 주사기의 플런저를 사용하여 50 ML 튜브에 나일론 메쉬 (70 μm의 기공 크기)를 통해 세포 현탁액을 전달하십시오. </li><li> 20 ML 5 % 태아 소 혈청 (FBS) -PBS와 나일론 메쉬를 씻으십시오. </li><li> 실온 (18 ° C)에서 7 분 (300 XG) 동안 원심 분리기. 흡인으로 상징액을 버리고 다음 단계로 진행하십시오. 뜨는 물을 제거 할 때 매우 조심하십시오. 흡인 피펫으로 쉽게 흡입 할 수있는 펠릿을 방해하지 마십시오. </li></ol> 4. 밀도 구배 <ol><li> 밀도 그래디언트 매체에 10x PBS의 적절한 금액을 추가하여 밀도 기울기 매체의 재고 솔루션을 준비합니다. 참고 : 한 부분의 10X PBS를 9 부분 밀도 그 레이디 언트 매체에 추가하십시오. </li><li> 표 1에 설명 된대로 다른 비율에 대한 1x PBS로 주식 밀도 그래디언트 매체를 희석하십시오. </li><li> 4 ML 30 % 밀도 그래디언트 매체와 펠렛 (3.7 단계에서)을 Resuspend하고 15 ML 튜브로 전송할 수 있습니다. </li><li> 15 ML 튜브의 바닥에 파스퇴르 피펫을 놓고 천천히 37 % 밀도 그라디언트 매체의 4 ML. </li><li> 위에서 설명한대로 70 % 밀도 그라디언트 매체 4 ML을 추가합니다. </li><li> 실온 (18 ° C)에서 40 분 (800 XG) 동안 그라디언트를 원심 분리하십시오. 계기가 방해받지 않도록 원심 분리기가 최소 또는 전혀 브레이크가 걸리지 않도록하십시오. 참고 : 튜브가 계층화되어 나타납니다. 70 % -37 % 밀도 구배 계면에서 적층 된 세포는 단일핵화 된 면역 세포 (소교 세포 및 대 식세포 하위 집단); 상위 수준은 세포 파편과 myelin입니다 ( 그림 2 ). </li><li&gt; 전송 피펫을 사용하여 myelin의 층을 부드럽게 제거하십시오. </li><li> 깨끗한 15 ML 튜브에 macrophage subpopulations 세포를 포함하는 70-37 % 밀도 그라디언트 interphase 3-4 ML을 수집합니다. </li><li> PBS 15ml의 총 볼륨으로 세포를 3x 씻으십시오. 세포가 들어있는 밀도 구배 매체가 PBS로 적어도 세 번 희석되었는지 확인하고 &quot;on&quot;브레이크로 7 분 (500 xg, 4 ° C) 동안 원심 분리하십시오. </li><li> 세포 라벨 또는 다른 기능 assays 1 ML PBS로 세포 펠렛을 Resuspend. </li></ol> 5. 유동 세포 계측법을위한 세포 라벨링 <ol><li> 플로우 cytometry 튜브에 세포 (단계 4.10)를 전송합니다. </li><li> 단계 4.10에서 얻은 각 세포 샘플에 고칠 수있는 죽은 세포 얼룩 시약 1 μL를 추가합니다. 빛으로부터 보호 된 얼음에서 30 분 동안 품어 낸다. </li><li> 5 분 (300 XG, 4 ° C) 2 ML PBS와 원심 분리기로 한 번 세포를 씻으십시오. </li><li> 뜨는을 제거하고 세포 펠렛을400 μL PBS 1 % BSA, 0.1 % 아 지드. </li><li> 1 μg CD16 / CD32 항체를 100 μL의 세포에 첨가하여 Fc 수용체를 차단하십시오. 얼음에서 10-15 분 동안 인큐베이션하여 빛으로부터 보호합니다. </li><li> 세포 내 얼룩에 대한 세포 permeabilization에 대한 PBS 1 % BSA, 0.1 % azide 또는 PBS 1 % BSA, 0.1 % azide, 0.25 % 사포닌의 기본 항체 믹스를 준비합니다. </li><li> 1 차 항체 100 μL를 넣고 (2 배) 얼음에서 30 분 동안 인큐베이션하여 빛으로부터 보호합니다. 참고 : 최적의 결과를 얻으려면 실험을 수행하기 전에 모든 항체를 적정해야합니다. </li><li> 5 분 (300 XG, 4 ° C) 2 ML PBS와 원심 분리기로 세포를 씻으십시오. 이 단계를 세 번 반복하십시오. </li><li> 일차 항체가 fluorophore에 직접 접합되지 않은 경우 보조 항체 100 μL를 추가하고 빛으로부터 보호 된 얼음에서 30 분 동안 품어 둡니다. </li><li> 4시 300 XG에서 5 분 2 ML PBS와 원심 분리기로 세포를 씻으십시오 ° C). 이 단계를 세 번 반복하십시오. </li><li> 수정빛으로부터 보호 된 실온에서 15 분 동안 100 μL 1 % PFA가있는 세포. 참고 :주의 : PFA는 유독합니다. 흄 후드에 넣고 개인 보호 장비를 착용하십시오. </li><li> 4시 300 XG에서 5 분 PBS 2 ML 및 원심 분리기와 세포를 씻으십시오 ° C. 이 단계를 세 번 반복하십시오. </li><li> 200 μL PBS로 세포 펠렛을 Resuspend 및 흐름 cytometry 수집 및 분석 진행합니다. 그림 3 의 게이팅 전략을 따른다. </li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> J Neuroinflammation. 9, 147 (2012).</li><li>Mizutani, 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=The+fractalkine+receptor+but+not+CCR2+is+present+on+microglia+from+embryonic+development+throughout+adulthood.">The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.</a> J Immunol. 188 (1), 29-36 (2012).</li><li>Lecoeur, H., Ledru, E., Gougeon, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytofluorometric+method+for+the+simultaneous+detection+of+both+intracellular+and+surface+antigens+of+apoptotic+peripheral+lymphocytes.">A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.</a> J Immunol Methods. 217 (1-2), 11-26 (1998).</li><li>Richter, N., 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=Glioma-associated+microglia+and+macrophages/monocytes+display+distinct+electrophysiological+properties+and+do+not+communicate+via+gap+junctions.">Glioma-associated microglia and macrophages/monocytes display distinct electrophysiological properties and do not communicate via gap junctions.</a> Neurosci Lett. 583, 130-135 (2014).</li><li>Mahad, 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=Modulating+CCR2+and+CCL2+at+the+blood-brain+barrier:+relevance+for+multiple+sclerosis+pathogenesis.">Modulating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis.</a> Brain. 129 (Pt 1), 212-223 (2006).</li></ol>

저자는 아무 것도 공개하지 않는다.

microglia와 MDM은 중추 신경계에서 서로 다른 기능과 표현형을 가지고 있으며 따라서 대 식세포 subpopulation의 확인과 분석은 신경계 질환 9 , 18 , 25 를 더 잘 이해하기 위해 필수적이라는 것이 입증되었습니다. 두 마커 (CD11b 및 CD45)를 사용하여 유동 세포 계측법 분석은 각 하위 집단 사이의 구별을 허용합니다 ( 그?...

미세 아교 세포는 중추 신경계 (CNS)의 실질 조직 내성 대 식세포입니다. 그들은 두 가지 중요한 기능적 역할을합니다 : 면역 방어와 CNS 항상성 유지. 골수에서 조혈 줄기 세포로부터 계속적으로 재생되는 MDM과 달리, 소구 세포는 배아 발달 중 뇌를 식민지로 만든 난황낭 (YS)에서 기원하는 조혈 전구 세포와 구별된다. 설치류에서 전사 인자 Myb는 모든 골수 유래 단핵 세포와 대 식세포의 발달에 중요한 역할을하지만, YS 유도 된 미세 아교 세포에 대해서는이 인자가 필요없고 분화는 전사 인자 PU.14에 의존한다. 건강한 CNS에서, 미세 아교 세포는 끊임없이 그들의 환경을 채취하는 역동적 인 세포입니다. sca침입하는 병원체 또는 조직 손상에 대한 육안 검사 및 측량 5 . 이러한 신호의 검출은 상해를 해결하기위한 경로를 시작합니다. microglia 신속하게 ramified 형태에서 amoeboid 하나, 다음 phagocytosis 및 프로 또는 항 염증성 cytokines 등 다양한 중재자의 릴리스 스위치. 따라서, 그들의 microenvironment에 따라, 활성화 된 microglia는 스펙트럼의 별개 프라이밍 상태 6을 얻을 수 있습니다. microglia는 근본적으로 많은 신경 장애의 발달과 진행에 영향을 미칩니다. 알츠하이머 병 (AD) 7 , 근 위축성 측삭 경화증 (ALS) 8 , 다발성 경화증 (MS) 9 또는 파킨슨 병 (PD) 10 의 설치류 모델에서, 소아 글 리아는 해로운 신경 독성 또는 행동을 유도하는 이중 역할을하는 것으로 나타났다 신경 보호적인 방식으로,n 특정 질병, 병기 및 질병이 전신 면역 부 7 , 8 , 9 , 10 , 11의 영향을 받았는지 여부. 상기 언급 된 질병에서 관찰 된 대부분의 중추 신경계 병변은 실질 세포 미세 소견뿐만 아니라 혈관 주위 및 수막 상피 세포뿐만 아니라 중추 신경계 침윤성 MDM을 포함하는 골수 세포의 이종 집단을 포함한다. 이러한 세포 유형은 손상 및 수리와 관련된 병리학 적 기전에 차별적으로 기여할 수있다 ( 7 , 12 , 13 , 14 , 15) . 이러한 질병 모델을 연구하는 연구자가 현재 직면 한 문제는 말초 단핵 세포와 대 식세포가 CNS에 침투하는지 여부를 확인하는 것이고, 그렇다면 dist이 세포에서 상주 microglia를 잉태하십시오. 실제로, 소교 세포는 매우 플라스틱입니다. 그들이 활성화되면, microglia는 일반적으로 말초 monocytes 및 macrophages에 의해 표현되는 마커를 다시 표현합니다. 따라서이 문제는 상주 소아 글로불린과 침윤 단핵구 및 대 식세포를 구분할 수있는 마커를 확인하는 데 달려있다. 면역 조직 학적 적용에 의한 뇌 조각에 대한 이들 집단의 차별은 특이 항체가 부족하기 때문에 제한적이다. 그러나, 유동 세포 계측법 분석은 여러 마커의 발현을 평가하고 세포 집단 (예 : 림프구, 대 식세포 / MDM CD11b + CD45 high 및 microglia CD11b + CD45 med )을 구별하는 효율적인 기술이며 세포 아군 16 , 17 , 18 . 이 프로토콜은최적화 된 효소 조직 해리 및 밀도 구배 원심 분리를 사용하여 신경계 질환 모델에서 마우스 CNS 유래의 단핵구; 뿐만 아니라, 유동 세포 계측법을 사용하여 중추 신경계에서 미 글로리아 및 MDM 모집단을 구별하는 방법을 제공한다. 또 다른 접근법은 특정 항체 19 , 20 , 21에 접합 된 ​​자기 구슬을 사용하여 미엘린을 제거하고 세포를 정제하는 것입니다. 안티 - myelin 자기 구슬을 사용하여 myelin 제거는 더 비싸며 분리 된 세포의 생존력과 수율에 영향을 미칩니다 22 . 이 단계와 microglia의 다음 immunomagnetic 분리는 특정 면역 세포 인구의 추가 연구를 제한 21 , 22 . 이 절차는 질병 발달에서 대 식세포 하위 집단을 연구하는 쉬운 방법을 제공하며,대 식세포 표현형 및 활성화 상태에 대한 약물 효과 또는 유전자 변형을 결정한다.

<table><tbody><tr><td>5 month-old Mice</td><td>Janvier</td><td>C57BL/6J</td><td></td></tr><tr><td>Liberase TL Research Grade</td><td>Sigma-Aldrich</td><td>5401020001</td><td>Digestion enzyme</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare Life Sciences</td><td>17-0891-01</td><td>Density gradient medium</td></tr><tr><td>Cell Strainer size 70 &amp;micro;m Nylon</td><td>Corning</td><td>731751</td><td></td></tr><tr><td>Venofix 25 G</td><td>BRAUN</td><td>4056370</td><td></td></tr><tr><td>Piston syringe 10 mL</td><td>Terumo</td><td>SS+10ES1</td><td></td></tr><tr><td>Pasteur pipette 230 mm</td><td>Dustcher</td><td>20420</td><td></td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030 123.328</td><td></td></tr><tr><td>15 mL tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>50 mL tube</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube</td><td>BD Falcon</td><td>352054</td><td></td></tr><tr><td>D-PBS (1x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>D-PBS (10x) without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>Bovine Serum Albumin solution 30%</td><td>Sigma-Aldrich</td><td>A7284</td><td></td></tr><tr><td>Paraformaldehyde 32% Solution</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Caution -Toxic</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>PerCPCy5.5 Rat anti-mouse CD11b (clone M1/70)</td><td>eBioscience</td><td>45-0112</td><td></td></tr><tr><td>Rat IgG2b K Isotype Control PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-4031</td><td></td></tr><tr><td>BV421 Rat anti-mouse CD45 (clone 30-F11)</td><td>BD Biosciences</td><td>563890</td><td></td></tr><tr><td>BV421 Rat IgG2b, &amp;kappa; Isotype Control RUO</td><td>BD Biosciences</td><td>562603</td><td></td></tr><tr><td>Rabbit anti-mouse TMEM119 (clone28 - 3)</td><td>Abcam</td><td>ab209064</td><td></td></tr><tr><td>AlexaFluor 647 Donkey anti-rabbit IgG</td><td>Life Technologies</td><td>A31573</td><td></td></tr><tr><td>Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Mouse Fc Block</td></tr><tr><td>Fixable Dead Cell Stain Kits</td><td>Invitrogen</td><td>L34969</td><td></td></tr><tr><td>Mouse CCR2 APC-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5538A</td><td></td></tr><tr><td>Rat IgG2B APC-conjugated Isotype Control</td><td>R&amp;amp;D</td><td>IC013A</td><td></td></tr><tr><td>Mouse CX3CR1 PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>FAB5825P</td><td></td></tr><tr><td>Goat IgG PE-conjugated Antibody</td><td>R&amp;amp;D</td><td>IC108P</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Cell analyzer</td><td>BD Biosciences</td><td>BD FACSVERSE</td><td></td></tr><tr><td>Data Analysis Software</td><td>FlowJo LLC</td><td>FlowJo</td><td></td></tr><tr><td>Fine scissors</td><td>F.S.T</td><td>14090-11</td><td></td></tr><tr><td>Standard Pattern Forceps</td><td>F.S.T</td><td>11000-13</td><td></td></tr><tr><td>Mayo Scissors</td><td>F.S.T</td><td>14010-15</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>F.S.T</td><td>11251-20</td><td></td></tr></tbody></table>

analysis of microglia and monocyte-derived macrophages from the central nervous system by flow cytometry

수많은 연구가 중추 신경계 (CNS) 병리학에서 면역 세포, 특히 대 식세포의 역할을 입증했다. 중추 신경계에 두 가지 주요 대 식세포 개체군이있다 : (i) 중추 신경계의 상주 대 식세포이며 배아 발생 과정에서 난황낭 전구 세포에서 유래 된 미세 아교 세포 및 (ii) 단핵구에서 유래 된 대 식세포 (MDM) 질병 중 중추 신경계는 골수 선조 세포에서 유래한다. 각 macrophage subpopulation의 역할은 연구중인 병리학에 따라 다릅니다. 또한, 이러한 macrophage subpopulation에 사용되는 histological 마커 또는 구분 기준에 대한 합의가 없습니다. 그러나, 유동 세포 계측법에 의한 CD11b 및 CD45 마커의 발현 프로파일의 분석은 우리가 MDM (CD11b + CD45 high )으로부터 미 글 리아 (CD11b + CD45 med )를 구별 할 수있게한다. 이 프로토콜에서는 밀도 구배 원심 분리유동 세포 계측법 분석은이 CNS 대식 세포 하위 집단을 특성화하고 최근에 출판 한 것처럼 이들 세포가 나타내는 여러 마커를 연구하는데 사용될 수 있습니다. 따라서,이 기술은 신경 질환의 마우스 모델에서 대 식세포의 역할에 대한 우리의 이해를 더할 수 있으며, 또한 이들 세포에 대한 약물 효과를 평가하는데 사용될 수있다.

밀도 구배 원심 분리 및 항체 염색 후, 세포를 유동 세포 계측기 (flow cytometer)상에서 획득하고 형태 학적 게이팅 전략을 사용하여하기와 같이 분석 하였다. 첫 번째 게이트는 이중 세포 ( 그림 3A )에서 단일 세포를 구별하기 위해 전방 산란 영역 (FSC-A) 대 전방 산란 - 높이 (FSC-H)로 정의되었다. 그런 다음 단일 세포를 FSC-A 대 Side Scattered-Area...

Watch this Scientific Journal Video about Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry at JoVE.com

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

이 프로토콜은 유행 세포 계측법에 의한 성체 마우스 중추 신경계에서 대 식세포 모집단의 분석을 제공하며, 이들 세포에 의해 표현되는 다중 마커의 연구에 도움이됩니다.

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 ...

analysis-microglia-monocyte-derived-macrophages-from-central-nervous

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Immunology and Infection

23.7K 조회수.  Inserm U 1227, CNRS UMR 7225.  이 프로토콜은 유행 세포 계측법에 의한 성체 마우스 중추 신경계에서 대 식세포 모집단의 분석을 제공하며, 이들 세포에 의해 표현되는 다중 마커의 연구에 도움이됩니다. 

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

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

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

일반 CNS의 실시간 PCR에 의한과 Neuroinflammation 동안 Microglia에 MicroRNAs의 탐지

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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 ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

단일 좌 골 신경 및 Cytometry 사용 하 여 단일 마우스에서 지 루트 신경 절에 면역 세포의 분석

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

신경과학

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

단일 핵 세포세포의 다중 파라미터 흐름 세포측정 분석을 위한 마우스 눈 전체의 소화

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

유세포분석을 사용한 마우스 뇌 히스톤 변형 정량화

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

저자 스포트라이트: 유전자 발현 조절 연구의 개선 사항

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

분리된 마우스 뇌 미세아교세포에서 핵내 유세포 분석을 사용한 글로벌 히스톤 번역 후 변형의 정량화

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

Percoll 그라디언트를 사용하여 두뇌와 척수 Mononuclear 세포의 분리

일반 CNS의 실시간 PCR에 의한과 Neuroinflammation 동안 Microglia에 MicroRNAs의 탐지

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

망막 미세 아교 식세포의 기능을 평가

단일 좌 골 신경 및 Cytometry 사용 하 여 단일 마우스에서 지 루트 신경 절에 면역 세포의 분석

단일 핵 세포세포의 다중 파라미터 흐름 세포측정 분석을 위한 마우스 눈 전체의 소화

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

분리된 마우스 뇌 미세아교세포에서 핵내 유세포 분석을 사용한 글로벌 히스톤 번역 후 변형의 정량화

분리된 마우스 뇌 미세아교세포에서 핵내 유세포 분석을 사용한 글로벌 히스톤 번역 후 변형의 정량화