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 (그?...

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.

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

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

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

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

림프구와 손쥐 Peyer의 패치에서 수지상 세포를 분리하고 Immunostaining

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

연구

면역학 및 감염병학

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 ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

3 차원 선 동적인 인간 조직 Gingiva 등가물

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

발생학

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

로컬 및 전신 사이트에서 병원체에 의한 만성 염증을위한 마우스 모델

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNA의 타겟팅 디곡신 표지 DNA 프로브를 사용하여 파라핀 조직 내에서 박테리아의 제자리 검출

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

숙주 세포와 혐기성 세균의 상호 작용을 평가하기위한 모델 생물로 포피 진지 발리

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

마우스와 인간의 치아에서 단일 세포의 신속한 격리

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

Y-27632를 사용하여 1차 인간 칭기발 상피 세포의 격리 및 배양

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

생체공학

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

편도선 단핵 세포의 격리는 인간 점막 림프구 조직에서 Ex Vivo 타고난 면역 반응을 연구합니다

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

In vitro에서 숙주-병원체 상호 작용을 조사하기 위한 유기형 조직 모델 시스템

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...