Bu çalışma Hibe R15 CA137499-01 (FB) ve Ohio Üniversitesi (FB) bir başlangıç ​​fonu altında NIH tarafından kısmen desteklenmektedir.

1. Fare femur ve kemik iliği hücrelerinin süresince tibialar ve kültür diseksiyonu <ol><li> CO 2 boğulma 2 fareler Kurban ve dikkatli bir şekilde kemik uçları kesmeden tibiaları ve femur teşrih. </li><li> Kağıt mendil kullanarak, bağlı tüm dokulardan kemikleri temizleyin. Kemikleri kırmak için değil dikkatli olun. </li><li> 35 mm Petri kabında 10 dakika boyunca% 70 etanol daldırma kemikleri sterilize edin. Bu andan itibaren, hücre kültürleri kirlenmesini önlemek için bir biyogüvenlik kaput içinde çalışır. </li><li> Etanol kemikleri kurtarma ve biyogüvenlik kabini içinde bir Petri kabındaki 5 dakika boyunca onları hava kurumaya bırakın. </li><li> En ince ucu yarısında femur ve tibia kesin. Steril bir Petri kabı steril bir şırınga kullanarak RPMI orta 1 ml (serum olmadan ancak antibiyotiklerle) ile kemik içinde infüze. </li><li> Hücre süspansiyonu sallanan bir kova rotor ile soğutmalı santrifüj 1100 RPM (4 ° C) 15 ml santrifüj tüpüne 10 dakika santrifüj RPMI ortamda toplanan ve 2X yıkanır. </li><li> Son yıkamadan sonra, ACK lizis tamponu 2 ml hücrelerin tekrar süspansiyon ve kırmızı kan hücreleri ortadan kaldırmak için oda sıcaklığında 5 dakika inkübe edin. </li><li> % 10 FBS RPMI 13 ml ekleyin, tekrar süspansiyon ve 1.6 &#39;da açıklandığı gibi aynı ayarları ile bu ortamda 2X yıkayın. </li><li> 2 ayarlamak, hücre sayımı RPMI% 10 FBS ve eklemek rm-GM-CSF (20 ng / ml son konsantrasyon) 5 x 10 5 hücre / ml. </li><li> CO 2 inkübatör (37 ° C,% 5 CO 2), steril, mikrobiyolojik kalite, 10 cm Petri kabı ve kültür için 10 ml süspansiyon ekleyin . </li><li> Üç gün sonra, hazırlanan plakaların her biri için 20 ng / l ile rmGM-CSF RPMI% 10 FBS başka bir 10 ml ekleyin. </li><li> Üç gün sonra 10 ml hücre süspansiyonu, her Petri kabı kurtarıldı 1.6 olarak santrifüj, rmGM-CSF RPMI% 10 FBS aynı hacimde yeniden süspanse ve Petri kabı geri döndü. Hücreler, CO 2 inkübatör 2 ek gün boyunca kültür . </li></ol> 2. DC&#39;ler Toplama ve etiketlenmesi <ol><li> 8 gün sonra kültür gevşek yapışık hücrelere taze orta Petri kapları yıkanarak kurtarıldı. Bu protokol elimizde 2-4 x10 yaklaşık 8 DC&#39;ler vermektedir. Hücreler flow sitometri DC fenotip için analiz edilebilir. </li><li> Toplanan hücreler daha sonra üreticinin talimatlarına sıkı sıkıya takip Qtracker 655 Hücre Etiketleme Kiti ile etiketlenir. </li><li> Biz 10 nM konsantrasyonda Qdots fare kemik iliği kökenli DH&#39;leridir etiketleme mükemmel sonuçlar aldı. Bunu başarmak için, aynı hacimde Qtracker Hücre Etiketleme Kit bileşenleri A ve B, eşit hacimlerde karıştırıldığında, oda sıcaklığında 5 dakika inkübe ve hemen 30 saniye vorteks taze orta 1 / 100 sulandırılmış. </li><li> 5 x 10 6 DC&#39;ler leke için, her kiti bileşenleri Eppendorf tüp ve oda sıcaklığında 5 dakika inkübe A ve B 5 ul karıştırın. Daha sonra, 1 ml RPMI% 10 FBS inkübe ve 30 saniye için vorteks ekleyin. </li><li> Içeren hücre süspansiyonu 5 x10 6 DC&#39;ler 0,5 ml ekleyin ve 37 inkübe ° C, 60 dk . </li><li> Daha sonra, RPMI% 10 FBS (1,100 RPM) 2X hücreleri yıkayın. Hücreler, deneysel fareler enjeksiyon için daha fazla kültür veya RPMI PBS içinde yeniden süspanse edilebilir. </li></ol> 3. Etiketli DC Değerlendirilmesi <ol><li> Hücre etiketleme floresan mikroskopi ile değerlendirilebilir. Bunu başarmak için, hücre süspansiyonu bir damla (Şekil 3, bir mikroskop lamı eklenir, bir cam lamel ile kaplı ve floresan mikroskobu bu Qdots sırasıyla 565nm ve 405-525nm emisyon ve eksitasyon spektrumları olduğunu dikkate alarak ile değerlendirildi. ) </li><li> Bu noktada, hayvanlar etiketli hücreler enjekte etmek için kullanılacak, ya da olgunlaşma bu hücrelerin 48 saat süreyle inkübe edilmesiyle tetiklenen olabilir (37 ° C, 5 CO 2) (100 ng / ml) ve TNF-(LPS varlığında 20 ng / ml). Floresan etiketleme DC olgunlaşma (Şekil 4) tarafından etkilenmez. </li></ol> 4. Temsilcisi Sonuçlar: Bu vesile ile biz fare kemik iliği kökenli DH&#39;leridir hazırlamak için nasıl tanımladı ve bunların nasıl floresan Qdots etiketli Şekil. Şekil 1, hücre elde etmek için yordamı özetlemektedir. 2 Qdots onları etiket prosedürü gösteriyor. Şekil. 3, neredeyse tüm hücreleri bu yordamı ile etiketlenir ve bu inflamatuar faktörlerin DC olgunlaşma (Şekil 4). Şekil etkilenmez. 5 inflamatuar bir kokteyl ile tedavi edildiklerinde Qdot lekeli DC&#39;ler (Fig.5a) lekeli olmayan hücrelere benzer davrandığını göstermektedir. Her iki hücre hazırlıkları kostimülatör moleküllerin benzer şekilde ifade upregüle (Şekil 5b); ve olgunlaşması uyaranlara üzerine IL-6 (Şekil 5c) ve nitrik oksit (Şekil 5d) benzer miktarlarda üretmek. Önceki teknolojik sıçramalarda ile aynı fikirdebu Qdot boyanma gösteren lı yayınlanan verilerin, immün yanıtları [6] eliciting yetenekli olgun hücrelere dönüşme yeteneği DC etkilemez. Bu hücreler daha sonra enjekte eden deney hayvanları için de kullanılabilir. Şekil. 6, farelere etiketli DC&#39;ler intravenöz enjeksiyon 2 gün sonra, biz, dalak Qdot lekeli hücreleri algılamak için başardık . Bu aynı zamanda, in vivo olarak DC göç değerlendirmek için Qdot nanokristallerin kullanımını gösteren yayınlanmış daha önceki veri ile anlaşma [6], non-invaziv görüntüleme ve akım sitometri. <img src="/files/ftp_upload/2785/2785fig1.jpg" alt="Şekil 1"/> Şekil 1 kemik iliğinden DC nesil akış şeması. Bu vesile ile biz, kemik iliğinden elde edilen DC elde süreci betimliyor. Tibiaları ve femur disseke ve doku çevresindeki temiz. Kemik ipuçları korunur ve kemiklerin iç vasatı ile yıka. , Kırmızı kan hücreleri ortadan kaldırarak sonra, kemik iliği hücreleri DC&#39;ler onları ayırt etmek için, GM-CSF huzurunda 8 gün boyunca kültür. <img src="/files/ftp_upload/2785/2785fig2.jpg" alt="Şekil 2"/> Şekil 2 etiketleme prosedürü akış şeması. Eşit alikotları Qtracker Hücre Etiketleme Kit bileşenleri A ve B Eppendorf tüp içinde karıştırılır. Dendritik hücreler, GM-CSF ile 8-gün kemik iliği kültürleri toplanan ve 37 ° C&#39;de 60 dakika etiketleme boya ile karıştırılır. Sonra hücrelerin aşırı etiketleme partikülleri ortadan kaldırmak için medya yıkanır. <img src="/files/ftp_upload/2785/2785fig3.jpg" alt="Şekil 3"/> Şekil 3 etiketleme hemen sonra DC&#39;ler Floresan microphotography. Etiketli hücreleri bir damla, bir cam slayt üzerine yatırılır ve bir lamel ile kaplıydı. Sonra floresan mikroskop, örnekler incelendi ve görüntüleri Micropublisher 5.0 Dijital CCD Renkli Kamera (Qimaging, Surrey, BC Kanada) yoluyla elde edildi. <img src="/files/ftp_upload/2785/2785fig4.jpg" alt="Şekil 4"/> Şekil 4 DC&#39;ler in vitro olarak 48 saat olgunlaşma sonra Floresan microphotography. Etiketli DC&#39;ler LPS (100 ng / ml) ve CO 2 inkübatör (37 ° C,% 5 CO 2) cam slaytlar TNF-α (20 ng / ml) ile 48 saat kültüre edildi. Daha sonra, örnekler, PBS (5 dakika, 2X) ile yıkanır aseton (15 dk, 4 ° C) ile sabit ve DAPI ile zıt. Hücreler, yukarıdaki gibi bir floresan mikroskop değerlendirildi. <img src="/files/ftp_upload/2785/2785fig5.jpg" alt="Şekil 5"/> Şekil 5 Qdot lekeli olgun DC&#39;ler Biyolojik aktivite. Yukarıdaki gibi in vitro olgunlaştı Etiketli DC&#39;ler flow sitometri analiz (A) ve (B). (A) Qdot boyanan hücrelerin FL3 (PercP) kanal güçlü bir sinyal verir. Gölgeli histogram: lekesiz kontrolü. Ve izotip kontrolleri, (B) Qdot lekeli ve lekesiz DC&#39;ler ayrıca CD86 ve CD40 olgunlaşma belirteçlerinin karşı spesifik antikorlar ile boyandı. Hücreler daha sonra bir FACSort akış sitometresi (Becton Dickinson, San Jose, CA) incelendi. Ayrıca, IL-6 (C) ve nitrik oksit (NO), olgun Qdot lekeli DC&#39;ler ve kontrollerin Süpernatantları tespit edildi. IL-6 ve nitrik oksit düzeyleri, ELISA analizi ve daha önce 7 açıklandığı gibi Griess tahlil, 8 sayesinde tespit edildi . Bu rakam gösterdi tüm veriler benzer sonuçlar gösteren iki ya da üç bağımsız deneyler temsilcisidir. Veri GraphPad yazılımı kullanılarak ANOVA ile analiz edildi. <img src="/files/ftp_upload/2785/2785fig6.jpg" alt="Şekil 6"/> Şekil 6 disseke dokularda etiketli DC&#39;ler tespiti. Etiketli olgun DC&#39;ler (1x10 6 hücre) intravenöz C57BL / 6 farelere enjekte edildi. (A) İki gün sonra fareler sakrifiye edildi ve toplanan dalak, dondurulmuş ek bileşenini, OCT gömülü ve 8 mcM bölümden bir Kriyostat kullanılarak hazırlanmıştır. Daha sonra, numunelerin aseton (15 dk, 4 ° C) sabit ve DAPI ile zıt. Hücreler yukarıdaki gibi bir floresan mikroskopta incelendi Şekil 6a: 40X büyütme, Şekil 6b: 100X büyütme, Şekil. 6C, 400X büyütme.

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T hücre etkileşimleri veya DC gelişimi ve çeşitli hastalıklar 9-11 rollerini belirlemek: DC araştırmak; murin miyeloid) DC&#39;ler yoğun DC-temelli aşıların etkinliği ve iyileştirme belirlemek amacıyla kullanılan edilmiştir. Bu vesile ile biz tibiaları ve femur kemik ilikleri kurtarıldı öncülerden DC&#39;ler oluşturmak nasıl göstermektedir. Biz, etanol 70% boğulma yolu ile bize bunları sterilize etmek için izin verir, böylece kontaminasyon olasılığını azaltmak ipuçları kesmeden kemikleri kurtarabilirsiniz. DC&#39;ler kemik iliği hücreleri ayırt etmek için sadece 5 Daha önce açıklandığı gibi GM-CSF kullanmak. Bazı protokoller de IL-4 kullanmalarına rağmen, GM-CSF (12) yüksek düzeyde çalışırken bu sitokin gerekli olmadığı bildirilmiştir. Gerçekten de, daha önce bu DC&#39;ler bağışıklık yanıtlarını 13 ikna etmek mümkün olduğunu göstermiştir. Ayrıca, bakım, bağlı hücreleri daha monosit gibi fenotip beri 8-günlük kültürlerden gelen orta ile Petri bulaşık sadece gevşek yapışık hücrelere kurtarmak için alınması gereken vardır. Burada DC&#39;ler floresan Qdot parçacıklar ile etiketleme göstermektedir. Bu etiketleme, diğer yöntemler açısından bazı avantajları vardır. İlk olarak, Qdots parçacıkları kolayca hücre içine dahil edilmiştir. İkincisi, floresan sinyal çok yüksek ve DC olgunlaşma değişmez. Üçüncü olarak, doku veya hücrelerin, aseton gibi çözücüler, GFP boyama protokolleri seçmek için şu anda daha fazla esneklik vererek, DC&#39;ler 14 etiketlemek için kullanılır ise ne olur aykırı ile sabit floresan kayıp değil . Son olarak, bu parçacıklar tarafından verilen yüksek floresan sinyal hücrelerin doku otomatik floresan rağmen görselleştirme sağlar. Daha önce 6 açıklandığı gibi, Qdot boyama, bu hücrelerin olgunlaşma yeteneği etkilemedi . Bu vesile ile biz Qdot-lekeli DC&#39;ler kostimülatör molekülleri upregulating ve inflamatuar uyaranlara yanıt olarak IL-6 ve nitrik oksit üreten, lekeli olmayan DC&#39;ler benzer bir şekilde davrandığını göstermektedir. DC&#39;ler bağışıklık tepkileri tetikleyen uzmanlaşmış hücreler olsalar da, kanser ve ateroskleroz 4, 15, 16 gibi patolojik durumların katılmak gösterilmiştir. Onlar da hatta yeni gemiler 19, 20 gelişmekte katılan yapısal olarak önerilen anjiyogenik süreci 17, 18, ​​katılmak için öne sürülmektedir. Böylece, DC in vivo izleme ve dokuların farklı 4, ​​21 coğrafi lokalizasyon belirlenmesi için izin yöntemleri, 22 çok değerlidir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> C57BL / 6 farelerinde </td><td> Jackson laboratuvarlar </td><td></td><td> Kadın, eski 6-8 hafta </td></tr><tr><td> RPMI </td><td> Invitrogen </td><td> 11875-119 </td><td></td></tr><tr><td> Fetal sığır serumu, nitelikli </td><td> Invitrogen </td><td> 10437-028 </td><td></td></tr><tr><td> Antibiyotik-antimikotik </td><td> Invitrogen </td><td> 15240-096 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 10010-049 </td><td></td></tr><tr><td> ACK Parçalama Tampon </td><td> Lonza Walkersville, Inc. </td><td> 10-548E </td><td></td></tr><tr><td> GM-CSF Rekombinant fare </td><td> PeproTech Inc. </td><td> 315-03 </td><td></td></tr><tr><td> Qtracker 655 Hücre Etiketleme Kiti </td><td> Invitrogen </td><td> Q25021MP </td><td></td></tr><tr><td> Lipopolisakkaritlerinin </td><td> Invivogen </td><td> tlrl-eblps </td><td></td></tr><tr><td> Rekombinant fare TNF alfa </td><td> Peprotech </td><td> 315-01A </td><td></td></tr><tr><td> CD86 antikor </td><td> BD Biosciences </td><td> 553691 </td><td></td></tr><tr><td> CD40 antikor </td><td> BD Biosciences </td><td> 553791 </td><td></td></tr><tr><td> Griess reaktif sistemi </td><td> Promega </td><td> G2930 </td><td></td></tr><tr><td> IL-6 yakalama antikor </td><td> eBioscience </td><td> 13-7061-81 </td><td></td></tr><tr><td> IL-6 algılama antikor </td><td> eBioscience </td><td> 13-7062-81 </td><td></td></tr></tbody></table>

generation and labeling of murine bone marrow-derived dendritic cells with qdot nanocrystals for tracking studies

Dendritik hücreler (DC), periferik dokularda ve immünolojik, timus, kemik iliği, dalak, lenf düğümleri ve Peyer Kullanıcı yamaları 1-3 gibi organlarda bulunan profesyonel antijen sunan hücreler (APC ). DC&#39;ler onlar, süreç ve major histokompatibilite moleküller (MHC) bağlamında kendi yüzeyinde bulunan antijenlerin varlığı, periferik dokularda örnek organizma mevcut. Sonra, antijen yüklü DC&#39;ler T lenfositlerin spesifik immün yanıtları tetikleyen işlenmiş antijenini immünolojik organlara göç ederler. DC&#39;ler göçmen yeteneklerini değerlendirmek için bir yolu, onları floresan boyalar 4 etiket . Bu vesile ile biz fare kemik iliği kaynaklı DC etiket Qdot floresan nanokristaller kullanımını göstermek. Bu etiketleme avantajı Qdot nanokristaller kurtarılan dokularda etiketli hücreleri tespit için ideal istikrarlı ve uzun ömürlü floresan sahip olmasıdır. Bunu başarmak için, ilk hücre DC farklılaşma ikna etmek için fare kemik ilikleri kurtarıldı ve granülosit makrofaj koloni uyarıcı faktör varlığında 8 gün kültüre olacaktır. Bu hücreler daha sonra in vitro inkübasyon kısa floresan Qdots ile etiketlenir. Boyanan hücrelerin floresan mikroskobu ile görüntülenebilir. Hücreler noktada deney hayvanlarının içine enjekte edilebilir ya da inflamatuar uyaranlara ile in vitro inkübasyon üzerine olgun hücrelerin içine olabilir Bizim ellerde, DC olgunlaşma floresan sinyal kaybı tespit etmedi ne Qdot boyama DC&#39;ler biyolojik özellikleri etkilemez. Enjeksiyon üzerine, bu hücrelerin tipik diseksiyon ve tespit işlemleri aşağıdaki floresan mikroskobu ile bağışıklık organlarda tespit edilebilir.

Watch this Scientific Journal Video about Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies at JoVE.com

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritik hücreler alımı antijenler ve T hücrelerine işlenir antijenleri bağışıklık organları doğru göç. Qdot nanokristal etiketleme, uzun ömürlü ve istikrarlı bir floresan sinyali sağlar. Bu floresan mikroskobu ile farklı organlara dendritik hücreler izleme sağlar.

İzleme Araştırmaları Qdot nanokristaller murin Kemik İliği-türevli dendritik hücrelerin üretilmesi ve Etiketleme

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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Research

JoVE Journal

Immunology and Infection

24.8K Görüntüleme.  Ohio University. Dendritik hücreler alımı antijenler ve T hücrelerine işlenir antijenleri bağışıklık organları doğru göç. Qdot nanokristal etiketleme, uzun ömürlü ve istikrarlı bir floresan sinyali sağlar. Bu floresan mikroskobu ile farklı organlara dendritik hücreler izleme sağlar.

Video: Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Kemik İliği Türetilmiş makrofajlar kullanarak Makrofaj Polarizasyon İncelenmesi

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

İmmünoloji ve Enfeksiyon

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Kemik İliği Makrofaj üretim kaynaklı

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Metabolik fenotipleri farklı olarak sahip Olgunlaşmamış, Olgun ve tolerojenik Dendritik Hücrelerinin Üretimi

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Fare Kemik İliği itibaren Üretimi ve GM-CSF tanımlanması Türetilmiş Alveoler gibi makrofajlar ve dendritik hücrelerin

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Çok sayıda miyeloid ataları ve dendritik hücre öncüleri fare kemik iliği strateji sıralama bir roman hücre kullanarak gelen nesil

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Vivo fare modeli ölçü saf CD4 T Hücre aktivasyonu, yayılması ve kemik iliği türevi dendritik hücreler tarafından indüklenen Th1 farklılaşma

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Kemik İliği Kaynaklı Dendritik Hücrelerin Farelerden İzole Edilmesi ve Kültürlenmesi için Ekonomik ve Verimli Protokol

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...