Name: isolation, processing and analysis of murine gingival cells
Uploaded: 2013-07-02T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

이 연구는 AW, AHH에 젊은 연구자 (GIF 영)에 대한 독일 이스라엘 재단과 박사 I에 이스라엘 과학 AHH로 재단 (제 11분의 1,418) 및 (제 12분의 1,933)에서 교부금에 의해 지원되었다 . AHH 및 AW에 치과 의학의 히브리어 대학 Hadassah 대학에서 Cabakoff 연구 기금 기금.

사전에 준비 : <ul><li> PBS + 2 % FCS </li><li> 콜라게나 유형 II와 DNase의 유형 I의 1 MG / ML (샘플 당 1 ML) 2 ㎎ / ml의 PBS + 2 % FCS </li><li> 멸균 수술기구 </li><li> 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 솔루션 </li></ul> 1. 위의 잇몸 절제 기법 <ol><li> 승인 IACUC 지침을 사용하여 쥐를 안락사. </li><li> 뺨과 날카로운 / 무딘 똑바로 가위로 하악 무스 등 구강의 양쪽을 잘라. </li><li> 하악을 아래로 당깁니다. </li><li> 표준 가위 분의 어금니 뒤에 가상의 선 1mm의 소프트 및 하드 조직을 잘라. 가위 구개 뼈 (그림 2A, 파란색 선)의 평면에 수직 연출합니다. </li><li> 소프트 및 하드 조직에게 앞니 (그림 2A, 파란색 선) 뒤에 2mm를 절개. </li><li> 입의 앞쪽 부분을 아래로 당깁니다. 비강이 단계 이후에 관찰된다. </li><li> 가위를 배치구개에 평행, 비강으로 한 잎을 넣어, 다른 날은 현관에서 절개한다. 후방 절개 (그림 2A, 녹색 선)까지 잘라냅니다. 상악의 양쪽에서이 단계를 반복합니다. 끝에서 상악은 두개골의 나머지 부분 (그림 2B)에서 분리된다. </li><li> , 상악를 제거 중간 봉합 (그림 2, 검은 점선)에 잘라 치조골에 도달 할 때까지 각 헤미 - 상악의 구개 조직을 잘라. </li><li> Adson 집게 (치아 제외)과의 앞쪽에 국경에서 치은 조직 (두 헤미 - 상악) 껍질. </li><li> PBS + 2 % FCS와 함께 접시에 절제 잇몸를 놓습니다. </li></ol> 2. 잇몸 처리 <ol><li> 1 ML PBS와 함께 조직 배양 접시 (35 × 10 ㎜)에 절개 잇몸을 넣고 + 2 % FCS, 콜라게나 유형 II와 DNase의 유형 I. 1 MG / ML 2 MG / ML </li><li> N ° 15 불임 수술 블레이드와 잘 조직을 말하다. </li><li> TR접시에서 15 ML 원뿔 시험관 조직을 ansfer. </li><li> 1 ML PBS + 2 % FCS와 동일한 테스트 튜브로 전송과 함께 접시에 남아있는 조직 / 세포를 씻으십시오. </li><li> 37 20 분 셰이커 인큐베이터에 품어 ° C, 200 rpm으로 (추천 : 예열 인큐베이터). </li><li> 37 또 다른 10 분 ° C, 200 rpm으로 20 개의 EDTA (에틸렌 다이아 민 테트라 초산) 0.5 M의 μL와 부화를 추가합니다. </li><li> 12 PBS + 2 % FCS와 ML 4에서 원심 분리기까지 테스트 튜브를 채울 ° C, 8 분 400 XG (추천 : 사전 냉기 원심 분리기). </li><li> 2 ML PBS + 2 % FCS로 뜨는 세포를 resuspend를 제거합니다. </li><li> 70 μm의 셀 스트레이너와 샘플을 필터링을 통해 흐름을 저장합니다. </li><li> 4 ° C, 5 분 320 XG에 원심 분리기. </li><li> 300 μL PBS + 2 % FCS로 뜨는 세포를 resuspend를 제거합니다. </li><li> 세포를 (약 5-10 × 10 5 세포는 단일 상악에서 예상되는) 계산. </li></ol> 3. 세포와 세포 내 항체유동 세포 계측법 분석을위한 염색 그것은 소등와 후드 내부에서 작업 할 것을 권장합니다. 그것은 지속적인 추운 환경에서 세포를 유지뿐만 아니라 필요한 모든 재료하는 것이 중요합니다. <ol><li> 100 μL의 총 부피 1 X 10 6 세포 당 선택한 각 항체의 0.2-0.5 μg을 추가하여 세포 분자에 세포를 얼룩. </li><li> 소용돌이 짧게. </li><li> 4 ° C.에 어둠 속에서 15 분 동안 튜브를 품어 </li><li> 2 ML PBS + 2 % FCS와 세포를 씻으십시오. </li><li> 4 ° C, 5 분 320 XG에 원심 분리기. </li><li> 추가 뜨는 세포를 resuspend를 대기음, 현명한 드롭, 500 μL 차가운 BD는 Cytoperm / Cytofix 소용돌이로 교반하면서. </li><li> 4 40 분 ° 어둠 속에서 C에 대한 튜브를 품어. </li><li> 소용돌이 짧게. </li><li> 1 ML 차가운 BD 파마로 두 번 세포를 씻으 / 4시 7 분 800 XG에 버퍼와 스핀을 씻으 ° C. </li><li> 300 μL를 제외하고 뜨는을 대기음. </li><li> 에 의해 세포 내에서 세포를 얼룩100 μL의 총 부피 1 X 10 6 세포 당 선택한 각 항체의 1 μg을 추가 할 수 있습니다. </li><li> 소용돌이 짧게. </li><li> 4 ° C.에 어둠 속에서 30 분 알을 품다 </li><li> 1 ML 차가운 BD 파마로 두 번 세포를 씻으 / 4시 7 분 800 XG에 버퍼와 스핀을 씻으 ° C. </li><li> 300 μL 차가운 BD 파마 / 워시 버퍼를 추가하여 상층 액과 세포를 resuspend을 대기음. </li><li> 소용돌이 짧게. </li><li> FACS 튜브 필터링을하는 동안 현탁액을 전송합니다. </li><li> 분석까지 어둡고 추운 환경에서 튜브를 유지합니다. </li></ol>

<ol>
	<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>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., 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=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

저자는 공개 아무것도 없어.

하나의 마우스에서 얻은 상악 치은 조직은 우리가 이전 1 설명한대로 세포와 세포 내 분자를 표현하는 T 및 B 림프구의 하위 인구뿐만 아니라, 자신의 능력을 분석하기에 충분합니다. 그럼에도 불구하고, 희소 한 세포 집단 (예를 수지상 세포에 대한) 관심이 있으며, 그것은 2-3 생쥐 수영장 조직에 권장됩니다. 참고로, 바람직하는 경우, 그것은 할 수 있습니다 다음, 구개 치은 조직 및 소비?...

잇몸은 치아의 치경부 부분을 둘러싸고있는 연부 조직과 폐포 과정 (그림 1)을 다룹니다. 치은 더 점막 상피 세포와 결합 조직 (또한 점막하 또는 고유 층라고도 함)로 분리 할 수​​ 있습니다 저작 점막의 유형입니다. 치은 및 인접 치아의 해부학 적 구조는 박테리아가 지속적으로 로컬 면역 체계를 도전 플라크 (바이오 필름)을 상주하고 발전 할 수 있습니다. 치주 질환 2를 칭했다 조건 - 결과적으로 염증 반응은 특정 상황에서 파괴되고 잇몸에 개발하고 있습니다. 기본적으로, 플라크에 의한 치주 병변은 치은염과 치주염으로 나눌 수 있습니다. 치은염은 잇몸에 국한되는 로컬 염증 반응의 가역적 인 상태를 나타냅니다. 치주염은, 다른 한편으로는, 돌이킬 수없는 파괴적인 과정이다하는 부착 장치 (치조골, 치주인대, 백악질과 치은은 3) 파괴된다. 잇몸이 치주 질환 4시 등의 이펙터와 유도 두 사이트를 제공하기 위해 제안되었다. 인간의 연구는 치과 플라크에 대한 응답으로, 면역 효과기 세포와 분자가 동적으로 침투하거나 출발 잇몸 5-7 것을 제안했습니다. 이 활동은 치주 파괴 8,9에 중요한 역할을하는 것으로 나타났다. 인간의 조직 작업이 병적 과정에 대한 귀중한 정보를 제공하는 연구에 의해 생성 된 데이터는 중요한, 윤리적 기술 및 실험 한계를 가지고 반면. 형질 전환 생쥐를 이용한 통해 생체 개입 10 인과 실험을 허용 실험 모델의 개발. 그 결과로, 치주 질환에 관여하는 메커니즘에 대한 우리의 지식은 지난 20 년간 크게 증가했다. 그럼에도 불구하고, 치주 질환의 복잡성으로 인해,이조직의 파괴를 촉진 지역 면역 반응의 특성에 관한 지속적인 논의. 치주 질환시 치은 중앙 면역 세포의 기능에 대한 이해의 부족이있다. 그것은 질병, 잇몸의 표적 조직에서 발생하는 병적 인 염증 이벤트를 공부 때문에 필수적이다.

<table><tbody><tr><td>Collagenase Type II</td><td>Worthington Biochemical Corp.</td><td>CLS-2</td><td></td></tr><tr><td>DNAse I</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>EDTA</td><td>SIGMA</td><td>E6758-100G</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>Biological Industries</td><td>04-121-1</td><td>Heat Inactivated</td></tr><tr><td>Vacuum-Driven Filter</td><td>Jet Biofil</td><td>FPE-204-500</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>SPL Lifesciences</td><td>93070</td><td></td></tr><tr><td>Tissue Culture Dish 35&amp;#215;10 mm</td><td>NUNC</td><td>153066</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>Anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD45.2 antibody</td><td>Biolegend</td><td>109819</td><td>Clone 104</td></tr><tr><td>Anti-mouse CD4 antibody</td><td>Biolegend</td><td>100413</td><td>Clone GK1.5</td></tr><tr><td>Anti-mouse CD8a antibody</td><td>Biolegend</td><td>100733</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>Biolegend</td><td>100214</td><td>Clone 17A2</td></tr><tr><td>Anti-mouse CD326 (Ep-CAM) antibody</td><td>Biolegend</td><td>118219</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse CD207 (Langerin) antibody</td><td>Imgenex</td><td>DDX0362D</td><td>Clone 929F3.01</td></tr><tr><td>Anti-mouse I-A&lt;sup&gt;d&lt;/sup&gt; (MHC-II) antobody</td><td>Biolegend</td><td>115010</td><td>Clone 39-10-8</td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo Software v 7.6.5</td><td>Tree Star</td><td></td><td></td></tr></tbody></table>

isolation, processing and analysis of murine gingival cells

우리는 정확하게 분리 및 유동 세포 계측법 및 분자 연구를위한 쥐 치은 조직을 처리하는 기술을 개발했습니다. 치은이 치주 질환을 일으킬 수 있습니다 구강 바이오 필름에 대한 숙주 면역 반응에 관여하기 때문에 면역 메커니즘을 연구하는 독특하고 중요한 조직이다. 또한 치조골 조직에 치은의 근접도 염증 상태에서 뼈의 리모델링을 공부 가능하게합니다. 우리의 방법은 우리가 이전에 1을 보여 같은 랑게르한스 세포와 T 조절 세포로도 희귀 한 세포 집단의 분석을 허용 면역 세포의 다량을 얻을 수 있습니다. 치주 질환시 치조골의 손실에 관련된 지역의 면역 반응을 연구하는 고용 쥐 때문에 다양한 면역 실험 도구의 가용성 유리합니다. 그럼에도 불구하고, 그들의 작은 크기와 쥐 잇몸에 상대적으로 불편 접근으로 인해 많은 연구가 생의 검사를 피할 수의 중요한 조직. 이 작품에서 설명하는 방법은 희망 치주 질환시 경구 면역 시스템과 그 역할을 우리 과소을 증가 잇몸 분석을 용이하게 할 수 있습니다.

잇몸 세포 유동 세포 계측법 분석의 예를 제시한다. 2 마우스에서 풀링 된 치은 세포 LSR II 흐름 cytometer에에서 실행 FlowJo 소프트웨어를 사용하여 분석 하였다. 그림 3A는 사이드 분산 형 (SSC) 대 앞으로 분산 형 (FSC) 플롯 순진 생쥐에서 치은 세포의 분포를 보여 주었다. 식별 할 수 게이팅 전략 (I) 림프구 (II) 단핵구 / 돌기 세포 그리고 (iii) 과립구가 표시됩니다. 비교 목적을 위해, 우리?...

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

이 연구는 단일 셀 문화를 생산하기 위해 마우스 구강의 잇몸 조직을 분리하고 처리 할 수​​있는 효율적인 방법을 설명합니다. 그 결과 세포는 더욱 유동 세포 계측법 분석 및 분자 연구에 사용할 수 있습니다.

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-processing-and-analysis-of-murine-gingival-cells

Research

JoVE Journal

Immunology and Infection

18.8K 조회수.  Hebrew University - Hadassah Medical Center. 이 연구는 단일 셀 문화를 생산하기 위해 마우스 구강의 잇몸 조직을 분리하고 처리 할 수​​있는 효율적인 방법을 설명합니다. 그 결과 세포는 더욱 유동 세포 계측법 분석 및 분자 연구에 사용할 수 있습니다.

비디오: Isolation, Processing and Analysis of Murine Gingival Cells

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.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

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 ...

연구

면역학 및 감염병학

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 ...

발생학

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

마우스 침샘에 줄기 세포의 분리

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

생물학

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

구강 호중구의 평가를 위한 뮤린 치주염의 강력한 합자 유도 모형

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

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

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

여러 중추 신경계 영역에서 쥐 아교 세포의 해부 및 분리

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

신경과학

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

쥐 관상 동맥 혈관 평활근 세포의 분리

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

성인 뮤 린 눈물과 턱 밑 동맥에서 Myoepithelial 세포의 고립

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...