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.

Bu protokol açıklanan tüm deneysel prosedürleri gerekli kurallar takip ve Kurumsal Hayvan Bakımı ve Kullanımı Komitesi, NIDCR / NIH tarafından kabul edildi. 1. Peşin hazırlayın <ol><li> Tam ortam hazırlayın: RPMI 2 mM L-glutamin, 100 birim / ml penisilin, 100 ug / ml streptomisin ve% 10 FBS ile takviye edilmiştir. </li><li> 7.5 ug DNaz ile desteklenmiş RPMI medya 50 ml (taze olun her zaman buz üzerinde tutmak): DNase ortamını hazırlayın. </li><li> DNaz medya artı 3.2 mg / kollajenaz Tipi IV ml 5 ml (taze olun her zaman buz üzerinde tutmak): kollajenaz-DNAse ortamı hazırlayın. </li><li> Ön-serin FACS tamponu: PBS içinde seyreltilmiş% 0.5 FBS. </li><li> 4 ° C&#39;ye ön-serin santrifüj. </li><li> 37 ° C&#39;ye ısınmaya çalkalayıcı inkübatör. </li></ol> Dişeti Blokları 2. İzolasyon, Diseksiyon ve Hücre İzolasyonu <ol><li> ARAC kurallarına göre uygun bir odasında CO 2 onları teşhir ederek fareler Euthanize. </li> &lt;li&gt; kalp ve iç organları ortaya çıkarmak için göğüs ve karın boşluğu açın. serpmek için, inferior vena kava bir kesi yapmak ve bir 27 G iğne ile 3 ml şırınga kullanarak sol karıncığın yoluyla PBS 3 ml serpmek. </li><li> yukarı bakacak şekilde mide ile bir pad üzerinde vücut ve fare kafasını hareketsiz. </li><li> altta yatan dokuları ve kasları açığa mandibula ve boyun bölgesini kapsayan cilt ayıran, makasla boyuna doğru dudak her komissura kesin. mandibulayı ortaya çıkarmak için alt dudak teşrih. </li><li> alt ön dişlerin arasına Cut mandibula her iki tarafını ayrı ve ağız boşluğu erişmek için her tarafı hareketsiz hale getirmek. </li><li> Görselleştirmek ve uzak burun boşluğundan damak teşrih. </li><li> 10 bıçak kullanarak alanları incelemek ve dişeti (Şekil 1) çevreleyen maksiller ve mandibular molar alanları bulunmaktadır. Dikey kesiler sadece birinci molar ve posterior üçüncü molar ve yatay kesi de için ön yapmakDişeti sınır (doku çevreleyen diş beyaz yaka). </li><li> 5 ml kollajenaz / DNaz (buz üzerinde tutmak) ile 50 ml konik tüp içinde çene ve alt çene blok yerleştirin. </li><li> 1 saat boyunca 37 ° C&#39;de çalkalamalı bir inkübatör içinde inkübe ve inkübasyon son 5 dakika boyunca 0.5 M EDTA, 50 ul ekle. </li><li> dokular ile 50 ml tüp soğuk DNaz medya 5 ml ekleyin ve hafifçe karıştırın girdap. </li><li> petri üzerinde doku 4 blok aktarın ve DNaz medya 500 ul ile kaplayın. Bir neşter bıçak kullanarak ve dişeti ve diş arası alanlara bıçak işaret ederek, doku her bloğunun gingiva çıkarın. </li><li> hücre süzgeç (70 mikron boyut) ile yeni bir 50 ml tüp, petri yanı sıra diseksiyonu sırasında kullanılan önceki DNaz medyadan dokuları ve medya aktarın. DNaz medya (3-5 ml) kullanılan araçların ve filtrenin tüm yıkayın. </li><li> steril 3 ml şırınga pistonu kullanarak, süzgecin karşı doku püreSoğuk DNase medya (30 ila 35 ml) ile filtreleme. </li><li> Soğuk DNaz medya ile hücre süzgeç yıkayın. </li><li> 4 ° C&#39;de santrifüj, 6 dakika boyunca 314 x g. </li><li> supernatant atmak eksiksiz bir medya 1 ml yeniden askıya. </li><li> Hücreleri saymak otomatik hücre sayacı kullanılarak (beklenen miktar&gt;% 80 canlılığı ile 6 10 x 1,2-5 10 x 8 arasındaki hücreleri arasında olmalıdır). </li></ol> Dişeti Hücrelerinin 3. stimülasyonu ve FACS Boyama <ol><li> Brefeldin A 1 ul / ml varlığında PMA (50 ng / ml) ve iyonomisin (2.5 ug / ml) ile tek bir hücre süspansiyonu teşvik </li><li> 37 ° C de 3.5 saat ve% 5 CO2 inkübe edin. </li><li> Spin ve 4 ° C&#39;de PBS ve santrifüj ile yıkayın; 6 dakika 314 x g. </li><li> Üreticinin talimatlarına göre bir canlılık leke kullanarak hücreleri leke. </li><li> dışı belirteçleri (CD45, TCRγδ, TCRβ, CD4, CD8, TCRγδ veya karınca için Lekeüreticinin talimatlarına aşağıdaki seçim ibodies). </li><li> 4 ° C&#39;de 5 dakika boyunca 314 xg&#39;de soğuk FACS tamponu ve santrifüj ile hücreler yıkanır. </li><li> Yavaşça hücre pelletini bozmaya girdap. </li><li> Oda sıcaklığında 20 dakika boyunca% 2 paraformaldehid hücreleri saptamak. </li><li> 4 ° C&#39;de, hücre içi sitokin 5 dakika ve leke 314 xg&#39;de soğuk FACS tamponu, santrifüj ile hücreler yıkanır. <ol><li> 4 ° C&#39;de 5 dakika boyunca 314 xg&#39;de FACS tamponu ve santrifüj içinde seyreltilmiş, soğuk% 0.5 saponin çözeltisi ile FACS tüpler doldurun. </li><li> FACS tüpler% 0.5 saponin çözeltisi 300 ul ekle, 4 ° C de karanlıkta 15 dakika boyunca inkübe edilir ve daha sonra 4 ° C&#39;de 5 dakika boyunca 314 x g&#39;de santrifüj. </li><li> imalatçının talimatlarına uygun olarak hücre içi işaretleri (IL-17A ve IFNy ya da tercih edilen sitokinler) için hücrelerin lekelenmesi. 5 dakika boyunca, 4 ° C&#39;de 314 xg% 0.5 saponin çözeltisi ve santrifüj ile hücreler yıkanır. </li><li> FACS ile tekrar yıkayıntampon ve 4 ° C&#39;de 5 dakika boyunca 314 x g&#39;de santrifüjleyin. </li><li> FACS tampon 300 ul hücrelerin yeniden askıya. </li></ol></li><li> Bir Akış sitometresindeki sonuçlarını analiz. </li></ol> 4. Akım Sitometri Analizi <ol><li> hücrelerin görselleştirmek enkaz dışlamak için ileri (FSC) ve yan dağılım (SCC) ve kapısı üzerinde analiz edilecek. </li><li> yüksekliği (FSC-H) parametreleri vs ileri dağılım alanı (FSC-A) Tek hücreleri seçin. </li><li> Daha fazla analiz (Şekil 2A ve 2C) için hematopoietik işaretleyici CD45 + için canlı (geçerlilik boyaması ile gösterildiği gibi) ve pozitif olan kapı hücreleri. </li><li> Şekil 2 ve Şekil 3&#39;te gösterildiği gibi, özel markörlerin analizi devam edin. </li></ol>

<ol>
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Authors have nothing to disclose

Mevcut teknik, başarılı bir şekilde uygun bir (tek bir fare), bağışıklık hücrelerinin büyük sayısını verir, sadece fenotipik karakterizasyon için değil, aynı zamanda fonksiyonel çalışmalar için ex vivo. Başka bir grup daha önce izole ve fare dişeti bağışıklık hücreleri karakterize bir protokol yayınlamıştır ve hayvan modellerinde 9 periodontitis çalışmada flow sitometri çok renkli kullanarak değerini tanıştırmıştı. Önemli sorun giderme Bu protokolde anah...

Gingival dokusu, insan ve murin diş yapısına çevreleyen sürekli diş 1 kompleks biyofilm maruz kalmaktadır. Dişeti bariyeri polislik bağışıklık hücre ağı patojenik meydan 2 karşı etkili bağışıklık sağlayan, aynı zamanda, doku bütünlüğünü korumak, yerel ortakçı mikroplarla homeostazisini sağlanması ve hayati önem taşımaktadır. Homeostazı elde etmek için, bağışıklık sistemi dikkatlice henüz küçük ayrıntı dişeti bağışıklık hücre popülasyonlarının ve doku bağışıklığı 2 korunmasında rolleri bilinen bir yüksek ihtisas bağışıklık hücre ağı oluşturma dişeti çevre için hazırlanmıştır. Immün homeostasisin gingiva de bozulduğu zaman, ya da daha büyük bir konakçı karşı duyarlılık ve / veya disbiyotik mikrobiyal topluluklar, enflamatuar bir durumun varlığı sayesinde, periodontit 3 4 ortaya çıkar. Periodontitis diş s kaybına yol açan yaygın bir inflamatuar bir hastalıktıraltlık yapılar. Onun ağır formlarında genel nüfusun 5 yaklaşık% 10&#39;unda görülür. Periodontitis duyarlılık ve ilerlemesinde rol önemli faktörler Kesme 6 zor olduğu kanıtlanmıştır. Ancak, hayvan modelleri periodontitis başlangıcında ve ilerlemesinde 7 mekanizmalarını anlamak son derece yararlı olmuştur. Modeller bağışıklık homeostazı ve periodontitis sürücü gelişimini sürdürmek için hayati önem taşımaktadır anahtar hücre popülasyonlarının ve moleküler mediatörler tanımlamak için kullanılabilir. Böyle bir anlayış bağışıklık homeostazı dişeti özgü kontrol anlayışımızı dönüşümü ve hastalık patogenezinde güncel anlamanız açısından çok önemlidir.

<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.

(- / - Şekil 2A - C LFA vs WT) protokolünün uygulanmasını göstermek için, bağışıklık hücresi ile ve periodontitis olmayan farelerin dişetinde ağı inceleyen temsili sonuçlar göstermektedir. Temsilcisi FACS araziler dişeti (Şekil 2A, 2C) canlı CD45 + hematopoetik hücreler gösterir. Bu protokol ile bağışıklık hücrelerinin izolasyonu ve işleme ex vivo uyarılması ...

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

Biz izolasyon, fenotipik karakterizasyonu ve kemirgen dişetinin gelen bağışıklık hücrelerinin fonksiyonel analiz için bir teknik kurduk.

İzolasyon, Karakterizasyonu ve Dişeti Bağışıklık Hücre Ağı Fonksiyonel Sınav

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.7K Görüntüleme.  NIH.  Biz izolasyon, fenotipik karakterizasyonu ve kemirgen dişetinin gelen bağışıklık hücrelerinin fonksiyonel analiz için bir teknik kurduk. 

Video: 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.

Lenfositler ve mürin Peyer en Yamalar itibaren Dendritik Hücreler İzolasyon Ve immün

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

İmmünoloji ve Enfeksiyon

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.

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

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.

Üç boyutlu inflamatuar insan dokusu jinjiva karşılıkları

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

Gelişim Biyolojisi

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.

Lokal ve sistemik Yer de patojen kaynaklı kronik enflamasyon Fare Modeli

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 rRNA Hedefleme Digoksin-etiketli DNA probu kullanılarak Parafine gömülmüş dokular içinde Bakterilerin in situ tespiti

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.

Ana Hücreleri ile Anaerobik Bakteriler Etkileşim değerlendirilmesi için bir model organizma olarak Porphyromonas gingivalis

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.

Fare ve İnsan Dişlerinden Tek Hücrelerin Hızlı İzolasyonu

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-27632 Kullanarak Birincil İnsan Dişeti Epitel Hücrelerinin İzolasyonu ve Kültürü

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

Biyomühendislik

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.

Bademcikler Mononükleer Hücrelerin İzolasyon Ex Vivo Doğuştan Gelen Bağışıklık Yanıtları Bir İnsan Mukozal Lenfoid Doku Çalışma

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.

Konak-Patojen Etkileşimlerinin İn Vitro Araştırılması için Organotipik Doku Model Sistemleri

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