Yazarlar, hücre sıralama ve Peter Mottram microphotographs ile yaptığı yardım için ona yardım için LSU Sitometrisi Core Tesisleri&#39;nde Marilyn Dietrich teşekkür etmek istiyorum. Bu çalışma, Uçuş Görevlisi Tıbbi Araştırma Enstitüsü, LSU-Rekabet Araştırmaları Programı Ödülü ve NIH / NIAID Hibeler P20 RR020159 ve R03AI081171 tarafından finanse edildi.

1. Akciğer perfüzyon ve tek bir hücre süspansiyonu <ol><li> Femoral ven yoluyla intraperitoneal ketamin / xylazine anestezik karışımı bir enjeksiyon (mg / fare ketamin 1.8, ksilizin 0.19) ve exsanguination fare Euthanize </li><li> Kesme ve periton dış deri hafifçe geri çekerek göğüs boşluğu Açığa. Diyafram, kalp ve akciğerlerin maruz göğüs kafesi keserek açmaya devam edin. </li><li> EDTA-HBSS (ücretsiz HBSS kalsiyum / magnezyum-1mm EDTA) ile dolu bir 5 ml şırınga kullanarak hafifçe akciğerler serpmek. 25 G iğne bağlayın ve sağ ventrikül içine iğne takın. Enjeksiyon sırasında sızıntıyı önlemek için iğne etrafındaki myocardiac doku forseps kullanarak güvenli. Sabit bir basınç korurken yavaşça enjekte çözüm. Doğru perfüzyon akciğer enflasyon ve pembe / beyaz bir renk değişimine neden olacaktır. </li><li> Bu doku herhangi bir potansiyel kontaminasyon atmak için lenf düğümlerine drene çıkarın. </li><li> Toplamakakciğer dokusu, buz üzerinde petri transferi, ve jilet kullanarak küçük parçalara doğrayın. </li><li> GentleMACS C tüpe 5 ml / akciğer kollajenaz sindirim çözümü içeren bir topraklı doku transferi (kollajenaz tip 1A 0.5 mg / ml artı tip IV sığır pankreas DNaz 20 mg / ml% 5 FBS, 100 U / ml penisilin ve 100 mikrogram içeren HBSS ml streptomisin) /. </li><li> GentleMACS dissociator tek bir hücre süspansiyonu elde etmek için kullanın. M_lung_01: program seçin. 30 dakika, 37 ° C&#39;de örnek inkübe edin. Tüp doku parçaları tekrar süspansiyon her 5 dakika çalkalayın. Program m_lung_02 ile devam edin . </li><li> 4 g 335 x 10 dakika için 15 ml konik bir tüp ve santrifüj örnek transfer ° C Bu adımdan sonra, tüm reaktifler ve santrüfüj tutmak, 4 ° C hücre canlılığı azalmayı önlemek için. </li><li> Süpernatant atın ve oda sıcaklığında 1 dakika için ACK parçalama tampon 2 ml ekleyerek kalan kırmızı kan hücreleri lyse. 13 hücreleri yıkayın4 335 x g &#39;de 10 dakika süreyle soğuk PBS/0.5% BSA ve santrifüj ° C ml </li><li> 5 ml soğuk PBS/0.5% BSA supernatant, tekrar süspansiyon toplam hücre atın ve 100 mikron naylon mesh geçirir. </li><li> Tripan mavi dışlama boya kullanılarak toplam hücre sayısını belirler. </li></ol> 2. Manyetik izolasyon ve CD11c + hücreleri zenginleştirme <ol><li> 4 ° C&#39;de 10 dakika boyunca tek bir hücre süspansiyonu 200 x g Santrifüj ve supernatant atın. </li><li> 10 8 toplam hücre başına 400 ul ayırma tampon yeniden süspanse hücre pelet (% 0.5 BSA, PBS ve 2 mM EDTA). 4 az 30 dakika süreyle anti-CD16/CD32 antikor (0.5μg / 10 6 hücreleri) kullanarak bağlama Blok Fc aracılı belirsiz antikor ° C </li><li> 4 200 x g &#39;de 10 dakika için 10 ml soğuk ayrılık tampon ve santrifüj hücre yıkayın ° C 400 ul ayırma tampon hücrelerin tekrar </li><li> 100 10 8 başına CD11c mikro-ul ekle </&gt; toplam hücre destek. İyice karıştırın ve 15 dakika buzdolabında (2-8 ° C) inkübe edin. </li><li> 4 ° C&#39;de 10 dakika boyunca 200 x g hücreleri yıkayın ve soğuk ayrılık tampon ve santrifüj 10 ml supernatant atın. </li><li> 3 ml soğuk ayırma tampon hücre pelletini tekrar. </li><li> AutoMACS Pro programı posseld seçerek TM Ayırıcı kullanarak manyetik ayırma gerçekleştirin. Olumlu bir kısmını (0,5 ml) toplayın. </li></ol> 3. Konvansiyonel dendritik hücre (CDC) izolasyon <ol><li> Anti-CD11c PE-Cy7 ve anti-IA/IE (MHC-II)-FITC antikorlar 30 dakika boyunca 4 ° C ile inkübe zenginleştirilmiş CD11c pozitif hücre süspansiyonu Optimize edilmiş antikor konsantrasyonu hücre 0.5 mikrogram / ​​10 6. </li><li> Hücreleri 4 335 x g &#39;de 10 dakika süreyle soğuk PBS/0.5% BSA ve santrifüj ° C ile yıkayın Süspanse edin PBS/0.5% BSA 0.5 ml hücre ve 40 mikron naylon örgü ile aktaracaktır. </li><li> Geçinsaflığı modunda FACS Aria hücre sıralayıcı kullanılarak hücrelerin sıralayabilirsiniz. Saflaştırılmış hücrelerin zarar görmesini önlemek için toplama tüpleri PBS/0.5% BSA 100 ul ekleyin. Hücrelerin hücre sıralama süreci boyunca soğuk hava sıcaklığında tutun. </li></ol> 4. Alternatif protokol, tek bir hücre süspansiyonu ve CD11c + hücreleri zenginleştirme elde etmek için <ol><li> GentleMACS dissociator değiştirilmesi, adım 1.5 topraklı akciğer dokusunda 37 ° C&#39;de 1 saat süreyle inkübe başı akciğer ve 5 ml kollajenaz çözüm içeren 15 ml konik boru transfer olabilir Vorteks hücreler her 15 dakikada bir doku parçaları tekrar süspansiyon için. Içinde ve dışında 20 G iğne bağlanan bir 3 ml şırınga ile örnek 6-8 kez geçerek doku bozabilir. Sürecinde kabarcıkları yapmaktan kaçının, aksi halde hücre canlılığı tehlikeye olacak. Tek hücre süspansiyonu elde edildikten sonra, 1.8 adım ile devam edin. </li><li> AutoMACSPro TM ayırma değiştirilmesitor, CD11c hücrelerinin zenginleşmesi için manyetik izolasyon, ayrıca hücre süspansiyonu geçen iki MACS sütun üzerinden elle yapılabilir. Uygun sütun seçimi, örneklem büyüklüğü olarak değişecektir. </li></ol> 5. Temsilcisi Sonuçlar: Akciğer CDC CD11c hi / MHC-II yüksek hücre popülasyonu olarak tanımlanır. Şekil. 1, CDC dalak (% 4.8) gibi bazı diğer dokulara kıyasla% 1.4 daha küçük bir yüzdesini temsil etmektedir. Ancak, dalak aksine, akciğer CD11c pozitif hücrelerin CDC daha farklı bir hücre popülasyonu da dahil olmak üzere, MHC-II farklı miktarlarda ifade. Tek hücreli bir ön hazırlıktan sonra, toplam akciğer hücreleri hücre verimi 3,8-3,0 biraz% 1 üzerinde% 60-70 oranında bir canlılığı x 10 7 hücre / akciğer CDC alt kümesi temsil edildi . Bu oran, toplam akciğer CDC, yaklaşık 10 ton artırılır CD11c manyetik izolasyon sonra arttıİmes orijinal hazırlama (Şekil 2A) (&gt;% 16). Ancak, MHC-II (CD11c hi / MHC-II lo, makrofajlar) düşük miktarda ifade CD11c hücreler akciğer CDC alt kümesi ile karışık bir yüksek kirlenmesine neden olan hücre popülasyonu (&gt;% 70) temsil etti. Şekil. 2B, CDC saflık&gt;% 96&#39;ya ulaşmış hücre sıralama adımdan sonra bu hücrelerin ortadan kaldırıldı. CDC tüm prosedürü sonra her zamanki verimi 5 x 10 4 hücre / akciğer oldu. Microphotographs tipik dendritler (üst panel) ile yuvarlak hücreler olarak CDC morfolojik özellikleri göstermektedir. Öte yandan, CD11c hi / MHC-II lo makrofajlar (MΦ, alt panel) 12 tipik hücresel protuberances gösterir morfolojik olarak farklı bir alt kümesi temsil etti . <img alt="Şekil 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> Şekil 1 fare akciğer ve dalakta Diferansiyel DC frekans. Akciğer birnd C57BL / 6 farelerin dalak doku kollajenaz ile toplanır ve tedavi edildi. Kollajenaz sindirim ardından, hücreler, anti-fare CD11c-PE-Cy7 ve anti-fare IA / IE-FITC ile boyandı. Temsilcisi akış sitometri araziler, akciğer ve dalakta CDC yüzdeleri (CD11c hi / MHC-II hi) göstermektedir . <img alt="Şekil 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> Şekil 2 fare akciğer CDC izolasyonu anti-CD11c mikro etiketli Fare akciğer tek hücre süspansiyonu ve otomatik hücre ayırıcı geçirilir. A) temsilcisi arsa CDC (CD11c hi / MHC-II hi) yüzdeleri gösterir ve Makrofajlar (MΦ MHC-II, CD11c hi / lo) CD11c zenginleştirme sonra nüfusu. Zenginleştirilmiş fraksiyonu daha fazla boyama, anti-fare CD11c-PE-Cy7 ve anti-fare IA / IE-FITC kullanılarak izledi. Çift pozitif hücrelerin sıralanır ve flow sitometri ile analiz edildi. IçinB) Temsilcisi araziler morfolojisi tespit hücreleri modifiye Wright-Giemsa boyama ile cytospun ve boyandı. CDC yüzdeleri ve hücre sıralama sonra MΦ göstermektedir. Microphotographs, akciğer CDC ve akciğer MΦ temsili bir resim göstermek için. Ölçek çubuğu = 20 mikron.

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Yazarlar ifşa etmek başka bir şey var.

Pulmoner fare DC İzolasyon solunum uyaranların geniş bir çalışma için önemli bir tekniktir. Bu hücrelerin elde edilmesi sürecinin yanı sıra hücre canlılığı ve saflık hücre kaybı önlemek kritik adımları içerir. Toplama önce akciğer perfüze sıra kirletici eritrositler azaltmak gibi herhangi bir periferik hücrelerini ortadan kaldırmak için yardımcı olacaktır. Akciğerlerin çok sayıda işlenir otomatik disosiasyon advanteogus olabilir, aksi takdirde Elle yapıldığında kollajenaz sindir...

<table width="200" border="1"><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> ACK parçalama tampon </td><td> Invitrogen </td><td> D6-0005DG </td><td></td></tr><tr><td> Anti-fare CD11c (HL3) </td><td> BD Pharmingen </td><td> 5580979 </td><td> PE-Cy7 konjuge </td></tr><tr><td> Anti-fare IA / IE (269) </td><td> BD-Pharmingen </td><td> 553623 </td><td> Konjuge FITC </td></tr><tr><td> Collangenase Tip 1A </td><td> Sigma </td><td> 9891-500mg </td><td></td></tr><tr><td> Hücre süzgeçler </td><td> BD Falcon </td><td> 352.340, 352.360 </td><td></td></tr><tr><td> CD11c (N418) mikro- </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNaz I </td><td> Sigma </td><td> D5025-150KU </td><td></td></tr><tr><td> Hank Dengeli Tuz çözüm </td><td> Invitrogen </td><td> 14170 </td><td></td></tr><tr><td> HEPES tamponu çözümü </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Petri kapları 60 mm </td><td> BD Falcon </td><td> 351016 </td><td></td></tr><tr><td> GentleMACS ™ C tüpler </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> Nazik MACS dissociator </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS-Pro ™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASCS Aria </td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

Akciğer dendritik hücreler (DC), solunum yolu kontrol tolerogenic tepkilerin yanı sıra 3 işgalci patojenlere 1,2 algılama temel bir rol oynar . Farelerde akciğer dendritik hücreler, en az üç ana alt kümelerini tarif edilmiştir: konvansiyonel DC (CDC) 4, plazmasitoid DC (PDC) 5 ve IFN-üreten katil DC (IKDC) 6,7. CDC altkümesi akciğer 8 en önemli DC alt kümesidir. DC alt kümeleri tanımlamak için bilinen ortak işaretleyici CD11c, tip I monositler, makrofajlar, nötrofiller ve bazı B hücreleri 9 da ifade edilir transmembran integrin (β2). Bazı dokularda, fare DC tanımlamak için bir işaretleyici olarak CD11c kullanarak en CD11c + hücreleri büyük doku uygunluk kompleksi sınıf II (MHC-II) yüksek düzeyde ifade eden CDC alt kümesini temsil eder, dalak, geçerlidir . Ancak, akciğer, bir daha heterojen dokuyan DC alt setlerini, MHC-II düşük seviyelerde dersin CD11c&#39;nin yüksek düzeyde ifade farklı bir hücre popülasyonu yüksek bir yüzdesi vardır. Karakterizasyonu ve çoğunlukla F4/80, dalak makrofaj işaretleyici ifade dayanarak, MHC-II CD11c hi lo akciğer hücre popülasyonu potansiyel bir DC 11 habercisi olarak, pulmoner makrofajlar daha yakın bir zamanda 10 olarak tespit edilmiştir. Fare PDC aksine, pulmoner immün yanıt CDC belirli rol çalışmada, bu hücrelerin izolasyonunda yardımcı olabilir belirli bir marker eksikliği nedeniyle sınırlı kalmıştır. Bu nedenle, bu çalışmada, yüksek derecede saflaştırılmış fare akciğer CDC izole etmek için bir prosedür açıklar. Pulmoner DC alt gruplarının izolasyon solunum patojenleri akciğer konağın immün yanıtı tetikleyebilecek çevresel faktörlerin yanı sıra tepki olarak bu hücrelerin fonksiyonu içgörüler kazanmak için çok yararlı bir araç temsil eder.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

Fare akciğer dendritik hücreler yüksek oranda saflaştırılmış açıklanmıştır. Özgül ağırlık, geleneksel dendritik hücre alt izolasyon verilir.

Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Research

JoVE Journal

Immunology and Infection

28.9K Görüntüleme.  Louisiana State University. Fare akciğer dendritik hücreler yüksek oranda saflaştırılmış açıklanmıştır. Özgül ağırlık, geleneksel dendritik hücre alt izolasyon verilir.

Video: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Fare Periton Boşluk Hücreleri İzolasyon

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

İmmünoloji ve Enfeksiyon

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Fare Barsak ayrı dendritik hücreler ve makrofajlar İzolasyonu ve Karakterizasyonu

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Fare Dendritik Hücre Alt Grupları İzolasyonunda bir Verimli ve Yüksek Verim Yöntem

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Dış Zar veziküller: İzolasyon ve Makrofajlar Onların Pro-inflamatuar Potansiyeli Analizi

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

İmünohistokimya ve Akım Sitometri Kullanımı Endobronşiyal Biyopsilerinde Mekansal Dağılımı ve Fenotipik Kimlik: İnsan Akciğer dendritik hücrelerin

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Hassas kesimli Canlı Pulmoner Dendritik Hücreler Visualize&#39;a Fare Akciğer Dilimleri

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

İnsan dişi üreme yolu dendritik hücreler yalıtım Phenotypical ve fonksiyonel çalışmaları için

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...