Yazarlar editoryal yardım için Anne Daron ve Pierre Louis Loyher teşekkür etmek istiyorum, iki-foton mikroskop ve yardım damızlık fareler için hayvan Facility &quot;NAC&quot; ve Camille Baudesson yardım için Plateforme Imagerie Pitié-Salpêtrière (PICPS). Hibe sözleşmesi kapsamında bu sonuçlara önde gelen araştırma Avrupa Topluluğu&#39;nun Yedinci Çerçeve Programı fon aldı (FP7 / 2007-2013) n 304.810 ° - baskınlar, ve n Université Pierre et Marie Curie gelen Inserm den 241.440-Endostem, ° &quot;Ortaya &quot;la gelen&quot; &quot;dan,&quot; Ligue contre le kanser Derneği la Recherche sur le Kanser dökün &quot;ve gelen&quot; Agence Nationale de la Recherche &quot;Program Doğuşu 2012 (ANR-EMMA-050). PH la &quot;Ligue contre le kanser&quot; tarafından desteklendi.

NOT: Tüm deney protokolleri Fransız Hayvan Deneyleri Etik Kurul tarafından onaylanan ve sayı A-75-2065 ile &quot;Hizmet Koruma et Santé Animales, Çevre&#39;den&quot; tarafından doğrulanmıştır. Örnek boyutları deneylerin tekrarlanabilirliğini sağlamak için seçilmiş ve hayvan etiği düzenleme 3R göredir. Mouse 1. Hazırlık <ol><li> Anestezi <ol><li> Görüntüleme kısa bir süre (en az 1 saat) için, ketamin (100 mg / kg) ve ketamin (10 mg / kg) ihtiva eden bir tuzlu su çözeltisi içinde 200 ul karın içinden enjeksiyonu ile fare uyuşturan. </li><li> Alternatif olarak, görüntüleme uzun süre (4 saat kadar), bir uyarlanmış maske aracılığıyla O 2 / N 2 O, bir 70/30 karışımı içinde buharlaşmış izofluran% 2.5 bir inhalasyon ile fare uyuşturan. </li><li> NOT: Bu şekilde daha kararlı bilinçsizlik sağlar ve ilaçların seri intraperitoneal önler. </li&gt; <li> Fare uyuşturulduktan sonra, pense ile ayak pedi uyarılması yoluyla bilinç için kontrol edin. </li></ol></li><li> Vasküler boyama / ek boyama <ol><li> Vasküler boyama için: kuyruk damarından damardan Rodamin-Dextran 200 ul (2 MDA salin tamponu içinde 10 mg / ml) enjekte edilir. </li><li> Nötrofil boyama için: kuyruk damarından salin çözeltisi 100 ul (klon 1A8) Ly6G-PE 2 ug enjekte edilir. </li></ol></li><li> Sabitleme <ol><li> Immobilizasyon için, mikroskop ve anestezik gaz İnhalatörde uyarlanmış bir ölçüye stereotaktik tutucu kullanın. NOT: sterotaktik tutucu hayvanın kafasını sıkmak için diğer çıkarılabilir ve uyarlanabilir iki metal plaka parantez, bir sabit olmak ve bir metal destek olduğunu. </li><li> Nefes hareketleri izolasyonu sağlamak için tespit plakaları arasında fare kafasını takın. Istinat plakaları ve sk germek için kafa arasındaki kulaklarını sıkıniçinde. istikrarın ve hayvan nefes refahı için kontrol edin. </li></ol></li><li> Saç derisini çıkarın <ol><li> Dikkatle gözlerle temas kaçınarak, steril bir aplikatör ile kafa derisi saç ıslak etanol% 70 kullanın. </li><li> Tam frontal kemik parietal kemiğin geriye doğru derisi kaldırmak için steril makas ve pense ile cilt kesin. Kapsamlı kanamayı önlemek için gözleri ve kulakları 3 mm&#39;ye kadar uzak tutun. Kafatası (periost) kapsayan gevşek bağ dokusu çıkarın. Sıcak PBS-batırılmış kağıt havlu ile kalan saç yıkayın. </li></ol></li><li> Daldırma sistemi kurmak <ol><li> Doğrudan dağılımını kontrol etmek, küçük bir iğne kullanılarak kafatası cerrahi yapıştırıcı ile 18mm çapında bir lastik halka yapıştırın. (30 ul yeterli olmalıdır) sızmasını önlemek için kauçuk halka tüm çevresi boyunca tutkal. </li><li> PBS-batırılmış kağıt havlu ile halka altında son bir kez kafatası temizleyin ve sonra tam daldırma için 37 ° C PBS ekleyinkafatası. Göz kuruluğu önlemek için fare her göz NaCl% 0.9 solüsyonu veya belirli oftalmik merhem bir damla ekleyin. Amaç altında stereotaktik tutucu sürükleyin. </li><li> Yaklaşık doğrudan iletim yoluyla mikroskop oküler kullanılarak alanını yerleştirin. </li></ol></li></ol> 2. İki foton Görüntüleme Edinme <ol><li> Seçici 680 1.080 nm ayarlanabilir, NUR ışık 140fs darbeleri sağlayan Safir kristal lazer, lazer ve güç kontrolü için bir acousto-optik modülatör: Ti ile birleştiğinde bir multiphoton mikroskop kullanın. 2 dikroik aynalar (565 nm ve 690 nm), 565/610 ve 500/550 bant geçiren filtreler kombinasyonu ile (3 floresan kanalları aynı anda kayıt etkin) 3 harici olmayan descanned dedektörleri (NDD) içeren bir yapılandırmayı kullanın ve bir 20 (NA = 1) suya daldırma hedefi × bir plan apochromat ile 485 kısa geçiren filtre,. </li><li> ANAES iyi homeothermy izin vermek için, ısıtma odacığı ayarlamaanestezisi sağlanır fare., NOT: Sıcaklık daha iyi stabilite için oturumu görüntüleme önce 1 saat ayarlanmış olabilir. Bizim durumumuzda, 32 ° C anestezi fare optimal vücut ısısını (36-37 ° C) elde etmek için seçilmiştir ve 4 saat kadar muhafaza edilir. Bu sıcaklık, kullanılan (dijital sistemi bu noktada kontrol etmek için kullanılabilir) sisteme adapte edilebilir. Buna ek olarak, kullanılan ısıtma odası siyah / koyu ve dış ışık kirlenmesini önlemek için sistemde (hedefleri ve motorlu plaka) bir kısmını kapsamaktadır. </li><li> Sistemi açın <ol><li> Safir lazer, daha sonra tüm mikroskop sistemi: Ti açın. Toplama yazılımı başlatın. 870 nm lazer dalga boyu aralığını ayarlayın. Kayıt için uygun NDD seçin. NOT: Bizim durumumuzda, ikinci harmonik üretimi (SHG) 19 ve mavi floresan proteini (eCFP) 485 kısa geçiş filtresi sonra NDD tarafından tespit edilir. 500/550 bant geçiren filtre ve rodaminle dekstran t ile tespit edildikten sonra eCFP da NDD tarafından tespit edilirO NDD 565/610 bant geçiren filtre sonra. </li></ol></li><li> Satın alma ayarlarını yapma <ol><li> Yazılımın &quot;canlı kazanım&quot; görüntüleme komutunu kullanın ve mikroskop yön kontrolü kullanarak, eCFP ve Rodamin sinyali ile bir bölgeye odağı ayarlamak. En az foto-hasarı ile en iyi bir sinyal-gürültü oranı elde etmek için en az bir lazer güç. NOT: Kullanılan güç ayarı ilgi bölgenin derinliğine göre değişir. Genellikle AOM ayarları 15 mW civarında hedefi ötesinde bir lazer gücü temsil% 20 civarında, ayarlanır. Her NDD foto-ağartma ve fotoğraf zararı azaltmak için Proje Yönetim Ekipleri kazanç gücünü artırmak için daha iyidir. Tipik satın alma ayarları tek 1.58 us piksel bekleme süresi ile tarama ve 1.5 büyütme 512 x 512 piksel çözünürlük vardır. </li></ol></li><li> Ilgi Seç bölgeleri <ol><li> Kayıt parametreleri ayarlandıktan sonra, tekrar &quot;canlı&quot; satın alma komutunu kullanınve gerçek zamanlı olarak izlenmesi gereken bir arzu (x, y, z) alanını seçmek için doku anket. NOT: Genellikle, biz vasküler ve parankimal alanlar hem de dahil olmak üzere bir alan seçti. </li><li> &quot;Pozisyon&quot; yazılım komutu ile alan kaydedin. Not: Bu komut x, y, farklı uzak alanlarda z koordinatlarını ezberler. </li><li> Seçin ve aynı prosedürü kullanarak (3 kadar) ilgi ek alanları kayıt. </li><li> Seçilen büyütme ile gerekli hacme göre birinci hafızaya pozisyona &quot;z-yığın&quot; yazılım komutu ile 3D z-yığını ayarlayın. İlk derin konumunu ve daha sonra yüksek konumunu ezberleyin. Derinliği ile lazer gücünü Normale. NOT: Birden fazla eşzamanlı alan görüntüleme için, 12 mikron kalınlığında bir hacim elde etmek her alan için 3 mikron z-adımlarla ayrılan 5 dilim kazanır. Bu ayarlar, çalışılan hücre tipine uygun olabilir. </li></ol></li><li> Başlat edinimi <ol><li> Seçmek220; zaman serileri &quot;yazılım komutu ve zaman aralığı süresini ve döngü sayısını seçin. &quot;Denemeyi başlatmak&quot; yazılım komutu seçerek gerekli zaman atlamalı ve süresine göre zaman atlamalı görüntüleme başlayın. NOT: Bizim durumumuzda, biz 30 dakika gerçek zamanlı temsil 60 döngüleri sırasında bir hacim, her 30 sn kazanır. Daha küçük zaman atlamalı damardaki hızlı hücre haddeleme izlemek için gerçekleştirilebilir. Bu durumda ince z-yığın ve en fazla 2 farklı alanlarda aynı anda kaydedilebilir. </li><li> Set up düzenli olarak izlenmesi. <ol><li> Her zaman bilinçsiz olduğundan emin olmak için hayvan izlemek. NOT: bıyık veya hayvanın bacakları Çalkalamalı canlanma göstergeleri vardır. Hayvanın solunum sarsıntılı durumda, izofluran miktarı biraz azaltılabilir. Edinimi sırasında Güçlü doku sürüklenme fare canlanma bir göstergesidir. </li><li> Hedefi emin olunuz batırılır. 37 ° C PBS betwee yenileyinn iki satın almalar (30 dk). NOT: edinimi sırasında sinyal Aşamalı kaybolması PBS kaçak göstergesidir. </li></ol></li></ol></li><li> Prosedürün Sonu <ol><li> Gerekli videolar kaydedilir sonra, hızla resüsitasyon önce servikal dislokasyon ile hayvan euthanize. Not: yara iyileşmesi uzunlamasına görüntüleme gerçekleştirmek için sınırlandırır 24-48 saat içinde bir yara oluşmasına yol açar. </li></ol></li></ol> 3. Veri Analizi <ol><li> Drift düzeltme <ol><li> 3D Otomatik izleme için Imaris Bitplane yazılımını kullanın. </li><li> Imaris 3D görüntü dizisi açın. Seçin komutunu &quot;nokta&quot;, ve homojen alanda dağıtılan mümkünse, 4 farklı bağlantı noktaları en az her zaman noktalarında için manuel izleme (klik + shift) oluşturmak. </li><li> &quot;Düzenlemek parça&quot; sekmesinde &quot;Doğru sürüklenme&quot; komutunu kullanarak sürüklenme düzeltme uygulayın. </li><li> T edinO x düzeltme, y ve z özellikle kalite artefactual kararsız önlemek için. NOT: düzeltme değil tatmin edici değilse, diğer bağlantı noktalarını deneyin ya da analiz videoyu hariç. </li></ol></li><li> Hücre izleme <ol><li> Komutu &quot;Yeni noktalar eklemek&quot; kullanın ve algoritma ayarı &quot;zamanla iz noktalar&quot; seçeneğini seçin. Bir sonraki adıma gidin. </li><li> &quot;Kaynak kanalı&quot; komutunu kullanarak ilgi tüm kanalda ya otomatik hücre izleme prosedürü uygulayın. 10 mikron Set nesneleri (yani, hücreler) çapı. Bir sonraki adıma gidin. NOT: seçim ölçülen sinyalin nesneler ve özgünlüğü arasındaki ayrım bağlıdır. Kısacası, tüm kanalda otomatik izleme Saptanan sinyal ilgi nesneye özgü yalnızca mümkündür. SHG ve eCFP hem 485 kısa geçiş filtresi sonra NDD tarafından tespit Çünkü, izleme daha spesifik ve 5 sonra NDD tarafından kaydedilen eCFP sinyalini kullanarak etkili olacak00/550 bant geçiş filtreleri. Kümelenmiş veya yoğun paketlenmiş nesneler 3.1.2 adıma benzer bireysel nesnelerin manuel seçim gerektirir. </li><li> , &quot;Kalite&quot; filtre türünü seçin belirsiz nesnelerin izleme dışlamak için eşik ayarlayın. Bir sonraki adıma gidin. </li><li> İki noktalar arasındaki &quot;Max mesafe&quot; ve &quot;Boşluk boyutu&quot; doğru parametreleri ile &quot;Brown Algoritması Motion&quot; seçiniz. Parça nesil doğrulayın. </li><li> Parçalar otomatik olarak hesaplanır sonra, &quot;parça süresi&quot; filtre türünü seçin Tracks (4 kez puan temsil eden) daha kısa, 120 sn ortadan kaldırmak için eşiğini ayarlamak. </li><li> Zorunlu: otomatik izleme sonra, parça kalitesini kontrol edin. <ol><li> Nesneleri parça yolları takip kontrol etmek için zaman çubuğunu önüne sermek. , &quot;Düzenlemek&quot; sekmesinde bulunan iz noktası düzeltme seçenekleri kullanın değilse: &quot;delete&quot; komutu (herhangi bir iz s silmek sahte veya uygunsuz parçaları silinoint ayrı ayrı) &quot;kesmek&quot; komutunu kullanarak. Parça yolu süreksiz ise, eksik zaman noktaları (klik + shift) ekleyin belirli bir yolda tüm noktaları seçin ve kullanmak &quot;düzenlemek parça&quot; sekmesinde komutunu &quot;connect&quot;. </li></ol></li></ol></li><li> Niceleme parametreleri <ol><li> &quot;Tercihler&quot; komutu &quot;İstatistik&quot; sekmesini seçin listesinde ilgi dinamik parametrelerini seçin. Seçin komuta ve oluşturulan excel dosyasını kaydetmek &quot;dosyasına tüm istatistikleri verme&quot;. NOT: Çeşitli dinamik parametreler doğrudan böyle bir parça uzunlukları, anlık hız, ortalama hız gibi yazılım elde edilen ve doğruluk takip edilmektedir. Hareketlilik katsayısı 6, 20 tespit edilebilir ancak doğrudan yazılım tarafından sağlanan değil. </li><li> Her hücre için, zaman (Dt), her dönem için değiştirmelerin ortalamasını hesaplamak (30 sn zaman atlamalı film için, ¨t 30, 60, 90 sn, vb olacak).Zaman aralığının bir fonksiyonu olarak kare yerinden ortalamasını çizilir. Eğrinin doğrusal parçası eğiminin hesaplanması ile motilite katsayısı elde edilir. </li></ol></li></ol>

<ol>
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D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu Rev Immunol. 26, 585-626 (2008).</li><li>Yost, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

in vivo görüntüleme yönteminin kritik noktaları görüntüleme süresini maksimize etmek ve inflamatuar hücrelerin dinamiklerini etkileyebilecek bakteriyel kontaminasyon ve inflamasyon riskini en aza indirmek için odak istikrarı sağlamak için vardır. Cerrahi kemik iliği erişmek için minimal gerçekleştirilen gibi kafatası kemik iliği Görüntüleme bu amaçlarını izler. Steril malzeme ve antiseptik kullanımı hücre homeostazındaki pertürbasyon neden olabilecek enfeksiyon riskini s?...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

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			<td>2MDa, 10mg/mL, Vascular staining</td>
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			<td style="width:171px;">Queryo Medical</td>
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			<td>Surgical Glue, rubber ring fixation</td>
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			<td height="21" style="height:21px;width:297px;">Small gauge needles</td>
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			<td>Carl Zeiss</td>
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tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Fare kafatası yapısı intravital görüntüleme, kemik iliği fizyoloji çalışmak için iyi bir fırsat sunuyor. Ön-parietal alanı çevresinde ince olan kemik, kemiğin aşınma olmadan medulla nişler erişim elde etmek mümkündür. 1 MacBlue transgenik fare kafatası geniş bir 2D alanını temsil Şekil. kemik matriksi esas olarak SHG 19 tarafından kolayca tespit edilebilir kollajen oluşmaktadır. Rodaminle Dekstranın Enjeksiyon kemik iliği damar ağı lekeleri ...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Fare Kemik İliği monositlerin Takip<em&gt; İn Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K Görüntüleme.  Sorbonne Universités, UPMC Univ Paris 06, CR7. Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. 

Video: Tracking Mouse Bone Marrow Monocytes In Vivo

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

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

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 ...

İmmünoloji ve Enfeksiyon

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Kemik İliği Transplantasyonu tarafından Fare Kolit immün ve non-immün Hücreler Gen Ekspresyonu İşlevsel Rolleri Farklılaşan

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

İzolasyon, Arıtma ve İşlevsel Araştırmalar ve Kabul edilmiş Transfer deneyler için Fare Kemik İliği Nötrofiller Etiketlenmesi

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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 ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murin Hind Ekstremite Uzun Kemik Diseksiyon ve Kemik İliği İzolasyonu

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

için Femur Pencere Odası Modeli<em&gt; İn Vivo</emFare Kemik İliği içinde&gt; Hücre Takibi

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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 ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Kimlik ve yalıtım Oligopotent ve Lineage için kabul edilen miyeloid ataları fare kemik iliği dan

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

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 ...