Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

이 프로토콜에 설명 된 모든 실험 절차는 필요한 가이드 라인을 따라 기관 동물 관리 및 사용위원회, NIDCR / NIH에 의해 승인되었다. 1. 사전에 준비 <ol><li> 전체 미디어 준비 : RPMI가 2 mM L- 글루타민, 100 단위 / mL 페니실린, 100 μg / mL 스트렙토 마이신 및 10 % FBS가 보충. </li><li> 7.5 μg의 DNase의 보충 RPMI 미디어의 50 ㎖ (신선한 확인 항상 얼음에 보관) : DNase의 미디어를 준비합니다. </li><li> DNase의 미디어 플러스 3.2 밀리그램 / 콜라게나 유형 IV ml의 5 ㎖ (신선한 확인 항상 얼음에 보관) : 콜라게나-DNase의 미디어를 준비합니다. </li><li> 사전 멋진 FACS 버퍼 : PBS에 희석 0.5 % FBS. </li><li> 4 ° C에서 미리 냉각 원심 분리기. </li><li> 37 ° C까지 따뜻한 진탕 배양기. </li></ol> 잇몸 블록 2. 절연, 해부 및 세포 분리 <ol><li> ARAC 지침에 따라, 적절한 챔버 내에 2 CO에 노출하여 마우스를 안락사. </li> &lt;리&gt;는 마음과 내부 장기를 노출 흉부와 복강을 엽니 다. 관류하기 위해, 하대 정맥의 절개를하고, 27 G 바늘 3 ML의 주사기를 사용하여 좌심실을 통해 PBS의 3 ㎖을 관류. </li><li> 이 위를 향 위장 패드에 몸과 마우스의 머리를 고정. </li><li> 기본 조직과 근육을 노출, 하악과 목 영역을 커버하는 피부를 분리, 가위로 목을 향해 입술의 각각의 접합면을 잘라. 하악을 밝히기 위해 아랫 입술을 해부하다. </li><li> 하부 앞니 사이 절단하는 하악골의 양측을 분리하고 구강에 액세스하는 각각의면을 ​​고정한다. </li><li> 시각화 멀리 비강에서 미각을 해부하다. </li><li> 10 블레이드를 사용하여 영역을 해부 및 치은 (그림 1) 주변과 상악과 하악 몰 영역을 포함한다. 수직 절개 단지 첫 번째 대구치 후방 세 번째 몰에 수평 절개시에 전방 확인잇몸의 경계 (조직 주변 치아의 화이트 칼라). </li><li> 5 ml의 콜라게나 / DNase의 (얼음 유지)와 50 ML 원뿔 튜브에 상악과 하악 블록을 배치합니다. </li><li> 1 시간 동안 37 ℃에서 진탕 배양기에서 배양 및 배양의 마지막 5 분 동안 0.5 M EDTA의 50 μl를 추가합니다. </li><li> 조직에 50 ㎖ 튜브에 차가운 DNase의 미디어의 5 ML을 추가하고 부드럽게 섞어 소용돌이 친다. </li><li> 페트리 접시에 조직의 4 블록을 전송하고 DNase의 미디어의 500 μL로 커버. 메스 블레이드를 사용하여 치은과 치간 영역으로 블레이드를 가리키는 조직의 각 블록에서 잇몸을 제거합니다. </li><li> 셀 스트레이너 (70 ㎛의 크기)를 통해 새로운 50 ㎖ 튜브, 페트리 접시뿐만 아니라 해부시 사용 이전 DNase의 매체에서 조직 및 미디어 전송. DNase의 미디어 (3-5 ml)에 사용 된 악기와 필터의 모든 씻으시오. </li><li> 멸균 3 ML의 주사기의 플런저를 사용하여, 스트레이너에 조직을 매쉬차가운 DNase의 미디어 (30-35 ml)로 필터링. </li><li> 차가운 DNase의 미디어와 셀 스트레이너를 씻으십시오. </li><li> 4 ° C에서 원심 분리기, 6 분 동안 314 XG. </li><li> 상층 액을 버리고, 전체 미디어의 1 ㎖에 다시 일시 중지합니다. </li><li> 세포 카운트를 자동 세포 계수기를 사용하여 (예상 사용량은&gt; 80 %의 생존 6 10 1.2 × 5-10 × 8 세포 사이의 범위한다). </li></ol> 잇몸 세포의 3 자극 및 FACS 염색 <ol><li> Brefeldin A의 1 μL / ㎖의 존재하에 PMA (50 NG / ml) 및 이오 노마 이신 (2.5 μg의 / mL)로 단일 세포 현탁액을 자극 </li><li> 37 ° C에서 3.5 시간, 5 % CO 2 품어. </li><li> 스핀 다운, 4 ° C에서 PBS와 원심 분리기로 씻어; 6 분 동안 314 XG. </li><li> 제조업체의 지침에 따라 생존의 얼룩을 사용하여 세포를 얼룩. </li><li> 세포 마커 (CD45, TCRγδ, TCRβ, CD4, CD8, TCRγδ 또는 개미에 대한 얼룩제조업체의 지침에 따라 선택의 ibodies). </li><li> 4 ° C, 5 분 동안 314 XG에 차가운 FACS 버퍼와 원심 분리기와 세포를 씻으십시오. </li><li> 조심스럽게 세포 펠렛을 방해하는 소용돌이. </li><li> 실온에서 20 분 동안 2 % 파라 포름 알데히드로 세포를 고정한다. </li><li> 4 ° C, 세포 내 사이토 카인 5 분, 얼룩을위한 314 XG에 차가운 FACS 버퍼, 원심 분리기와 세포를 씻으십시오. <ol><li> 4 ° C, 5 분 동안 314 XG에 FACS 버퍼와 원심 분리기에 희석 차가운 0.5 % 사포닌의 솔루션을 FACS 튜브를 입력합니다. </li><li> FACS 튜브에 0.5 % 사포닌 용액 300 μl를 추가, 4 ° C에서 어둠 속에서 15 분 동안 배양 한 후 4 ° C, 5 분 동안 314 XG에 원심 분리기. </li><li> 제조업체의 지침에 따라 세포 내 마커 (IL-17A와 IFNγ 또는 선택의 사이토 카인)에 대한 세포를 얼룩. 5 분, 4 ° C에서 314 XG 0.5 % 사포닌 용액과 원심 분리기와 세포를 씻으십시오. </li><li> FACS 다시 세척버퍼 4 ° C, 5 분 동안 314 XG에 원심 분리기. </li><li> FACS 완충액 300 μL에 세포를 다시 일시. </li></ol></li><li> 유동 세포 계수기에 대한 결과를 분석합니다. </li></ol> 4. 유동 세포 계측법 분석 <ol><li> 세포를 시각화하는 파편을 제외 앞으로 (FSC)와 사이드 분산 (SCC) 및 게이트에서 분석한다. </li><li> 높이 (FSC-H) 매개 변수 대 앞으로 분산 형 지역 (FSC-A) 단일 셀을 선택합니다. </li><li> 추가 분석 (도 2A 및 2C)에 대한 조혈 마커 CD45 +의 라이브 (생존 염색법으로 표시됨) 및 양성 게이트 셀. </li><li> 도 2 및도 3에 도시 된 바와 같이 특정 마커의 분석을 계속한다. </li></ol>

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+The+Normal+Bacterial+Flora+Of+The+Oral+Cavity.">Defining The Normal Bacterial Flora Of The Oral Cavity.</a> J Clin Microbiol. 43, 5721-5732 (2005).</li><li>Belkaid, Y., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+And+Systemic+Control+Of+Tissue+Immunity+By+Commensals.">Compartmentalized And Systemic Control Of Tissue Immunity By Commensals.</a> Nat Immunol. 14, 646-653 (2013).</li><li>Darveau, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontitis:+A+Polymicrobial+Disruption+Of+Host+Homeostasis.">Periodontitis: A Polymicrobial Disruption Of Host Homeostasis.</a> Nat Rev Microbiol. 8, 481-490 (2010).</li><li>Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomicrobial+Pathogenesis+Of+Periodontitis:+Keystones,+Pathobionts,+And+Host+Response.">Immunomicrobial Pathogenesis Of Periodontitis: Keystones, Pathobionts, And Host Response.</a> Trends Immunol. 35, 3-11 (2014).</li><li>Eke, P. I., 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=Prevalence+Of+Periodontitis+In+Adults+In+The+United+States:+2009+And+2010.">Prevalence Of Periodontitis In Adults In The United States: 2009 And 2010.</a> J Dent Res. 91, 914-920 (2012).</li><li>Moutsopoulos, N. M., Lionakis, M. S., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+Errors+In+Immunity:+Unique+Natural+Models+To+Dissect+Oral+Immunity.">Inborn Errors In Immunity: Unique Natural Models To Dissect Oral Immunity.</a> J Dent Res. , (2015).</li><li>Hajishengallis, G., Lamont, R. J., Graves, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enduring+Importance+Of+Animal+Modelsin+Understanding+Periodontal+Disease.">The Enduring Importance Of Animal Modelsin Understanding Periodontal Disease.</a> Virulence. 6, 229-235 (2015).</li><li>Sharrow, S. O., Coligan, J. E., 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=Analysis+Of+Flow+Cytometry+Data.">Analysis Of Flow Cytometry Data.</a> Current Protocols In Immunology. , (2001).</li><li>Arizon, 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=Langerhans+Cells+Down-Regulate+Inflammation-Driven+Alveolar+Bone+Loss.">Langerhans Cells Down-Regulate Inflammation-Driven Alveolar Bone Loss.</a> Proc Natl Acad Sci U S A. 109, 7043-7048 (2012).</li><li>Moutsopoulos, N. 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=Defective+Neutrophil+Recruitment+In+Leukocyte+Adhesion+Deficiency+Type+I+Disease+Causes+Local+IL-17-Driven+Inflammatory+Bone+Loss.">Defective Neutrophil Recruitment In Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17-Driven Inflammatory Bone Loss.</a> Sci Transl Med. 6, 229-240 (2014).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-23-IL-17+Immune+Axis:+From+Mechanisms+To+Therapeutic+Testing.">The IL-23-IL-17 Immune Axis: From Mechanisms To Therapeutic Testing.</a> Nat Rev Immunol. 14, 585-600 (2014).</li></ol>

Authors have nothing to disclose

현재의 기술이 성공적으로 적합한 (단일 마우스)에서 면역 세포의 수가 산출뿐만 아니라 표현형 특성화뿐만 아니라 기능적 연구 생체. 다른 그룹은 이전에 분리하여 치은 뮤린 면역 세포를 특성화하는 프로토콜 번역 및 동물 모델 9 치주염의 연구에서 유세포 빛깔의 사용 가치를 도입했다. 상당한 문제 해결이 프로토콜의 주요 수정을 따르면 치은 칼라 영역과 치간 치은 (그림 1)를 모두 포함하기 위해 조직의 전체 블록의 해부를 포함했다. 단독으로 치은 박리 종종 전체 두께 플랩이 달성되지 않고, 치은의 일부 특히 치간 영역에서 누락된다. 이 방법을 사용하여 수확 된 면역 세포의 수는 크게 증가한다. 이 기술은 또한 치은 조직 상악의에서 모두 최적의 분리를 허용D 하악. 이 프로토콜의 중요한 단계는 치은 지역의 보수적 해부 및 해부을 효율적으로 속도를 포함한다. 특히, 조직의 블록 치아 (잇몸)의 주위에 최소한의 조직이 아니라 구강 조직을 포함하는 해부한다. 블록 신속하게 해부해야하며, 조직과 세포를 최적의 세포 생존을 보장하기 위해 얼음에 남아 있어야한다. 문제 해결을 목적으로 그 세포의 낮은 수율 콜라, 콜라게나 제 및 / 또는 조직 블록에서 치은 불충분 절개 부적절한 농도 배양 부적절한 시간에 관련 될 수 있음을 주목하는 것이 중요하다. 그 메모에서, 치은 사용 확대 돋보기 (2X-6X)의 해부는 도움이 될 수 있습니다. 세포의 생존 능력은 낮은 절차에서 미디어, 완충제 및 얼음에 계속 남아 있지 셀 지각과 관련 될 수있다. 조직 DIS의 유형에 내재이 프로토콜의 제한,sected 다른 해부학 배리어 연구 된 부위 (예. 피부 또는 위장관) 큰 조직을 사용할 수있는 비교 잇몸 조직의 크기이다. - / - 자발적으로 치주염을 개발 모델 그럼에도 불구하고, 여기에 제시된 기술은 우리가 LFA에서 지배적 인 세포 집단의 특성을 허용했다. LFA에서 - / - 마우스는 우리가 치주염 (10)의 개발 과정에서 지배적 인 IL-17 사이토 카인의 서명을 확인했다. 다색 유세포 분석을 이용하여, 우리는 IL-17 (11)의 셀 소스를 정의 할 수 있었다. IL-17의 설정이 질병에 로컬 TCRγδ 치은 (10) 내의 T 세포, CD4 + T 세포 및 선천성 림프 세포 (ILC)에 의해 제조 하였다. 이 방법에도 작은 개체군을 식별하는 능력을 가진 절연 셀 실질적인 번호, 지금 포괄적 이해 개발할 수있을 것이다치은 장벽의 무결성을 보호 전문 면역 세포 네트워크의 ING. 이 중요한 통찰력은 우리가 치은 환경에서 면역 반응을 평가 할 수 있습니다. 이 기술을 사용함으로써, 우리는 이제 추가적인 분석과 특성화를 수행 할 수 있도록, 유세포 셀 정렬하여 치은 면역 세포 망의 특정 구성 성분을 분리하기 위해 진행할 수있다.

치은 조직은 인간 및 뮤린 치열을 둘러싸고 끊임없이 티스 (1)의 복잡한 생물막에 노출된다. 치은 배리어 치안 면역 세포 망은 병원성이 챌린지에 대한 효과적인 면역성을 제공하는 동시에, 조직의 무결성을 유지하는 로컬 공생 미생물과 항상성을 보장하고 필수적이다. 항상성을 달성하기 위해, 면역 시스템은 신중 아직 작은 디테일이 치은 면역 세포군 및 조직 내성 (2)를 유지하는 역할로 알려진 고도로 전문화 면역 세포 망을 생성 치은 환경에 맞게 조정된다. 면역 항상성이 치은에 중단되면, 어느 증가 호스트 감수성 및 / 또는 dysbiotic 미생물 지역 사회, 염증 상태의 존재를 통해, 치주염 3 (4)를 발생한다. 치주염은 치아의 손실로 이어지는 일반적인 염증성 질환입니다upporting 구조. 그것의 심한 형태는 일반 인구 (5)의 약 10 %에서 볼 수있다. 치주염 감수성과 진행에 관여하는 중요한 요소를 해부하는 것은 6 어려운 입증했다. 그러나, 동물 모델은 치주염 개시 및 진행 (7)의 메커니즘을 이해하는데 매우 유용 하였다. 모델 면역 항상성 및 치주염의 구동 발전을 유지하는데 필수적인 주요 세포 집단과 분자 매개체를 정의하는데 이용 될 수있다. 이러한 통찰력은 면역 항상성의 치은 특정 제어에 대한 우리의 이해를 변환 및 질환 발병의 우리의 현재 이해를 촉진 할 것이다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fine Scissors</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">14058-11</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Handle #3</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Blades #10</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10010-00</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Splinter Forceps</td>
			<td style="width:188px;">Integra Miltex</td>
			<td style="width:188px;">6-304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Needles with regular bevel&#160;</td>
			<td style="width:188px;">BD Medical</td>
			<td style="width:188px;">305109</td>
			<td>27G, 12.7 mm length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Monoject syringes</td>
			<td style="width:188px;">Covidien</td>
			<td style="width:188px;">8881513934</td>
			<td>Luer-lock tip, 3mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">PBS, pH 7.4</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:188px;">10010-049</td>
			<td>Without Calcium and Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:188px;">Lonza</td>
			<td style="width:188px;">12-167F</td>
			<td>Without L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DNase I from bovine pancreas</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">DN25-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Collagenase type IV</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal Bovine Serum</td>
			<td style="width:188px;">Gemini Bio-products</td>
			<td style="width:188px;">100-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin 50 mg/ml</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">120-098-661EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Pen Strep</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">L-Glutamine</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA pH 8.0</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">351-027-721EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">50 mL tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352070</td>
			<td>Polypropylene, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Petri dishes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">351029</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">5 mL FACS tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352052</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD GolgiPlug</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:188px;">555029</td>
			<td>Contains brefeldin A solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Phorbol 12-Myristate 13-Acetate (PMA)</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ionomycin Calcium Salt</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">13909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Saponin from quillaja bark</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td style="width:188px;">Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD45 Alexa Fluor 700</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">56-0451-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD4 eFluor 450</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">48-0042-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse TCR beta APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse gamma delta TCR FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5711-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IL-17A APC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">12-7311-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IFN-&#947; PE</td>
			<td style="width:188px;">eBioscience</td>
			<td>11-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse NK1.1 PE-Cy7</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">25-5941-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD90.2 APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-0902-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD3e FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0031-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD19 FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0193-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11b FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0112-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11c FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0114-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse TCR beta FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6G FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6C FITC</td>
			<td style="width:188px;">BD Pharmingen</td>
			<td style="width:188px;">553104</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, characterization and functional examination of the gingival immune cell network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

(- / -, 그림 2A - C LFA 대 WT) 프로토콜의 응용 프로그램을 설명하기 위해, 우리는 면역 세포와 치주염이없는 마우스의 치은의 네트워크를 검사 대표적인 결과를 보여줍니다. 대표 FACS 플롯은 치은 (그림 2A, 2C)에 살고 CD45 + 조혈 세포를 표시합니다. 이 프로토콜로 면역 세포의 분리 처리가 생체 자극 사이토 카인 분비 패턴의 특성을 수행 할 수있는 세포를 산출한다. 여기에서 우리는 정상 상태 (WT) 총 CD45 + 세포에서 IL-17 및 IFN-γ 염색을 표시하고 마우스에서 (그림 2B - D) (인해 LFA 결핍) 치주염을 나타내는. 이러한 데이터는 조직 정상 상태 (WT) 동안 로컬 및 치주염과 관련하여 사이토 카인 정보를 캡처하기 위해이 기법의 전위 공개 (LFA - / -). 마이크에서E는 치주염에 민감 우리는 또한 특히 T 세포 및 선천성 림프 세포 (ILC)의 구획 (- B도 3a)의 치은 면역 세포 조성물을 특징으로한다. 우리 TCRβ + CD4 + T 세포에서 IL-17 및 IFN-γ의 생산을 정의 할 수 있었다 TCRβ + CD8 + T 세포, TCRγδ + T 세포, NK 및 ILC 구획 (도 3C). <img alt="그림 1" src="/files/ftp_upload/53736/53736fig1.jpg" /> 그림 1 :. 상악과 격리 된 치은 세그먼트 해부의 경계선을 보여주는 쥐 치은의 분리 그림은 잇몸 조직의 절개에 사용되는. 블록 해부 및 절연 블록에 대한 개요와 쥐 상악 (몰 지역)의 (B) 세그먼트. 치은과 상악 대구치 세그먼트의 헤 마톡 실린 및 에오신 염색 (H &amp; E)<html> L 로우 (C)에 도시되고 설명 된 조직 및 (D) 높은 배율. 원래 배율 지적했다. <a href="https://www.jove.com/files/ftp_upload/53736/53736fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53736/53736fig2.jpg" /> 그림 2 : 잇몸 세포에서 사이토 카인 생산을위한 대표 FACS 데이터. (A - C) FACS 플롯은 PMA와 생체 다시 자극 다음 마우스 치은 세포의 준비에 살고 CD45 + 세포 게이트를 보여주는 WT 및 LFA에 이오 노마 이신 - / -. (B - D) (인해 LFA-결핍) 치주염없이와 / 마우스의 잇몸에서 총 CD45 + 세포 마우스에서 IFN-γ와 IL-17 염색을 보여주는 대표 FACS 플롯을.세틸 / ftp_upload / 53736 / 53736fig2large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 3" src="/files/ftp_upload/53736/53736fig3.jpg" /> 그림 3 :. TCRγδ는 CD45 + 세포 게이트에 비해 염증이 잇몸에 면역 인구에 의한 사이토 카인 생산 (A) 대표 FACS 플롯 TCRβ에 대한 염색을 표시합니다. TCRβ + 세포 상에 게이팅 중간 플롯 TCRβ 염색 대는 CD4 (TCRβ + CD4 + T 세포, TCRβ + CD8 + T 세포 및 TCRγδ + T 세포의 분석을 가능)을 나타낸다. TCRγδ - - TCRβ에 게이팅 셀을 마우스 오른쪽 그래프는 NK1.1 염색 (NK 세포의 분석을 위해 수 있습니다) 대 CD45을 보여줍니다. (B) CD90.2에 대한 염색을 보여주는 대표 FACS 플롯은 CD45 + 혈통 음성 세포에 게이트 (CD3- CD19 - NK1.1 - 중 CD11c - CD11b를 - Ly6G - Ly6C - TCRγδ - TCRβ -)하는 타고난 림프 세포를 식별합니다. (C) 확인 된 세포 집단에서 IFN-γ와 IL-17 염색을 보여주는 대표 FACS 플롯. 치은 준비 20 주 이전 LFA에서입니다 - / - 치주염을 개발 마우스; 데이터는 3 개의 독립적 인 실험의 대표입니다. <a href="https://www.jove.com/files/ftp_upload/53736/53736fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

우리는 분리, 표현형 특성화 및 쥐의 잇몸에서 면역 세포의 기능 분석을위한 기술을 설립했다.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

isolation-characterization-functional-examination-gingival-immune

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K 조회수.  NIH.  우리는 분리, 표현형 특성화 및 쥐의 잇몸에서 면역 세포의 기능 분석을위한 기술을 설립했다. 

비디오: Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

심장 기능 면역 세포의 분리

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

쥐과 잇몸 세포의 분리, 처리 및 분석

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

지방 조직 면역 세포의 분리

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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 ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

절연 및 특성화 Neutrophil 파생 된 미의 기능 연구

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.