Name: selective harvesting of marginating-pulmonary leukocytes
Uploaded: 2016-03-11T15:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Yazarlar ve bu çalışma NIH / NCI hibe # (SBE kadar) R01CA172138 tarafından desteklendi.

Hayvan denekleri Prosedürleri Tel-Aviv Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Sıçan Protokolü <ol><li> hazırlıklar <ol><li> 2 kelebek 21 G iğne düzenleyin. İsteğe bağlı olarak, iğnenin keskin kenar dosyalama 2 künt kenarlı kelebek 21 G iğne hazırlamak. </li><li> yerçekimi (kuru) ayarında en az 30 dakika süreyle 121 ° C&#39;de otoklav ile sterilize makas 2 çift, körelmiş kenarlı forseps, hemostat, diş doku forseps: cerrahi aletler sterilize edin. </li><li> fosfat tamponlu tuzlu su (1 x PBS), çözeltinin ml&#39;si başına heparin 30 adet ekleyerek heparinize PBS (30 birim / ml) hazırlayın. Oda sıcaklığında kullanın. 35 ml hayvan başına ihtiyaç duyulan minimum hacimdir. </li><li> Hava kabarcıklarını önlemek için peristaltik pompa hatları ve kelebek iğne içine heparinize PBS akışı. </li></ol></li><li> MP-lökosit Akciğerler ve Toplama perfüzyon <ol><li> % 8 izofluran aşırı dozda ile hayvan Euthanize. izleme solunum durması ile ötenazi onaylayın. </li><li> Solunumun durması üzerine, derhal steril makas ve diş doku forseps kullanılarak periton ve göğüs boşlukları açın. kılıç şeklinde sürecine karın kadar birlikte orta hat karın kesisi gerçekleştirin. </li><li> herhangi bir iç organ ya da büyük kan damarları delmeden, dikkatli her iki tarafta göğüs kafesini kesme, forseps kullanarak sternum kaldırın. sternum üzerinde bir hemostat kelepçe ve rostrally kardiyopulmoner kompleksi ortaya çıkarmak için göğüs kafesi katlayın. </li><li> kalbin sağ ventrikül içine, bir kelebek iğne yerleştirin ve yaklaşık bir dakika içinde (~ 250 g hayvan 6 ml) bir şırınga içine kan maksimal hacim toplar. </li><li> Karaciğer (Tartışma) geriye doğru perfüzyon önlemek için hemostatla vena kava Kelepçe. </li><li> p çıkış borusu ile kan içeren şırınga değiştirirken sağ ventrikül içinde kelebek iğne tutuneristaltic pompa. </li><li> interventriküler septum nüfuz kaçınarak sol ventrikül içine, bir 5 mi hasat şırıngaya bağlanan ikinci bir kelebek iğne yerleştirin. </li><li> yaklaşık olarak 5 mL / dakikalık bir hızda bir peristaltik pompa açın ve yavaşça şırıngaya kan ile kirlenmiş olan perfüzat ilk mililitre toplar. perfüzat kırmızı renk soluk karanlıktan açılana kadar devam edin. (Kan bulaşmış perfuste atın). </li><li> peristaltik pompa kesilmesi olmadan, hızlı bir şekilde 20 mi hasat şırınga ile 5 mi hasat şırınga yerine ve perfüzyon daha yüksek bir hızda (kadar 7 ml / dakika) kullanılarak, akciğer perfüzat 20 ml toplar. Vakum oluşumunu kaçınarak sürekli toplama enjektöre perfüzyon akışını izlemek. </li><li> peristaltik pompa akışını kesmeye. </li></ol></li><li> lökositler Ekstraksiyon <ol><li> 400 x g&#39;de 10 dakika boyunca santrifüj perfüzat. </li><li> sıvıyı aspire. </li><li>10 dakika boyunca 400 x g&#39;de 10 ml PBS veya orta, santrifüj ekleyin ve sıvıyı aspire. İki kez bu adımı yineleyin. PBS veya ortam kullanılması numunesinin sonucu kullanımına bağlıdır. </li><li> 10 dakika boyunca 400 x g&#39;de 20 ml PBS veya orta, santrifüj ekleyin ve sıvıyı aspire. </li><li> Örnek kullanım (FACS PCR, vb) göre istenen konsantrasyona hücreleri sulandırın. </li></ol></li></ol> 2. Fare Protokolü <ol><li> hazırlıklar <ol><li> 2 kelebek 25 G iğne düzenleyin. İsteğe bağlı olarak, iğnenin keskin kenar dosyalama 2 künt kenarlı kelebek 25 G iğne hazırlamak. </li><li> yerçekimi (kuru) ayarında en az 30 dakika süreyle 121 ° C&#39;de otoklav ile sterilize makas 2 çift, körelmiş kenarlı forseps, hemostat, diş doku forseps: cerrahi aletler sterilize edin. </li><li> 1x PBS çözeltisi ml&#39;si başına heparin 30 adet ekleyerek heparinize PBS (30 birim / ml) hazırlayın. Oda sıcaklığında kullanın. 25 ml hayvan başına ihtiyaç duyulan minimum hacimdir. </li><li> streHava kabarcıklarını önlemek için peristaltik pompa hatları ve kelebek iğne içine PBS heparinize ediyorum. </li></ol></li><li> MP-lökosit Akciğerler ve Toplama perfüzyon <ol><li> % 8 izofluran aşırı dozda ile hayvan Euthanize. izleme solunum durması ile ötenazi onaylayın. </li><li> Solunumun durması üzerine, derhal steril makas ve diş doku forseps kullanılarak periton ve göğüs boşlukları açın. kılıç şeklinde sürecine karın kadar birlikte orta hat karın kesisi gerçekleştirin. </li><li> herhangi bir iç organ ya da büyük kan damarları delmeden, dikkatli her iki tarafta göğüs kafesini kesme, forseps kullanarak sternum kaldırın. sternum üzerinde bir hemostat kelepçe ve rostrally kardiyopulmoner kompleksi ortaya çıkarmak için göğüs kafesi katlayın. </li><li> Karaciğer (Tartışma) geriye doğru perfüzyon önlemek için hemostatla vena kava Kelepçe. </li><li> Sağ ven içine peristaltik pompa çıkış borusuna bağlı bir kelebek iğne yerleştirinKalbin tricle ve kalp, septum ve avoidingpenetration sol ventrikül içine, bir 2 mi hasat şırıngaya bağlanan ikinci bir kelebek iğne. </li><li> yaklaşık 2 ml / dk hızına pompa açın ve perfüzyon (kan bulaşmış perfuste atın) kırmızı soluk karanlıktan dönene kadar hasat şırıngaya kan kirlenmiş perfüzat ilk mililitre toplamak. </li><li> peristaltik pompa kesilmesi olmadan, hızlı bir şekilde 10 mi hasat şırınga ile 2 mi hasat şırınga yerine ve perfüzyon daha yüksek bir hızda (en fazla 4 ml / dakika) kullanılarak, akciğer perfüzat 10 ml toplar. Vakum oluşumunu kaçınarak sürekli toplama enjektöre perfüzyon akışını izlemek. </li><li> peristaltik pompa akışını kesmeye. </li></ol></li><li> lökositler Ekstraksiyon <ol><li> 400 x g&#39;de 10 dakika boyunca santrifüj perfüzat. </li><li> sıvıyı aspire. </li><li> 400 10 ml PBS veya orta, santrifüj ekle10 dakika xg ve sıvıyı aspire. Bu adımı üç kez tekrarlayın. PBS veya ortam kullanılması numunesinin sonucu kullanımına bağlıdır. </li><li> 10 dakika boyunca 400 x g&#39;de 10 ml PBS veya orta, santrifüj ekleyin ve sıvıyı aspire. </li><li> Örnek kullanım (FACS PCR, vb) göre istenen konsantrasyona hücreleri sulandırın. </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Burada sunulan yöntem seçici hasat sağlayan ve MP-lökositlerin eşsiz nüfusun eğitim. Döner ya da dalak lökositleri karşılaştırıldığında, MP popülasyonu bir lökosit alt gruplarının farklı bileşim, yüksek aktivasyon düzeyleri, muhtelif sitokinlerin yüksek bırakma ve yanlısı ve anti-enflamatuar sitokinlerin 2,3 daha yüksek mRNA seviyeleri ile karakterize edilir. Özellikle, MP-NK hücreleri, çeşitli hedef hücrelere 3,4 karşı dolaşım NK hücreleri daha sitotoksiktir...

Akciğer kılcal (örneğin, marginating pulmoner (MP) lökositler) 1 diğer bağışıklık bölümlerinde (örneğin, kan dolaşımı, karaciğer, dalak, kemik iliği) gelen lökositlere kıyasla belirgin lökosit bileşim ve özel aktivitesi sergiledikleri gösterilmiştir ve 2- yapışan lökositlerin 4. Örneğin, dolaşım ve dalak NK hücreleri, hem de ayırt mesajcı RNA (mRNA) seviyeleri ve pro- ve anti-enflamatuar sitokinlerin artan salgılanmasında karşılaştırıldığında çeşitli tümör hücrelerine karşı MP-lökosit sergi yüksek doğal katil (NK) hücreler sitotoksisite. MP-lökosit dolaşan lökositlerin (% 50 vs sırasıyla% 30,) ile karşılaştırıldığında doğal / adaptif bağışıklığın daha yüksek bir oranına sahip olarak hücrelerin kompozisyonu da dolaşan lökositlerin ayırt edilir. Burada sunulan yöntemin amacı, MP-lökositlerin selektif hasat sağlamak, bu önemli ve eşsiz bağışıklık bölmesi (hücre popülasyonu) incelemek için, ve eluc için iseBu özel hücreler üzerinde çeşitli manipülasyonlar (örneğin, bağışıklık aktivasyon) etkisini idate. Bu eşsiz nüfusun önemini anlamak için, bağışıklık sisteminin hücre-aracılı bağışıklık (CMI) in vivo fonksiyonları ile tümör hücrelerini, mikrometastazlar ve rezidüel hastalığın dolaşan kontrol edebilirsiniz dikkat etmek önemlidir. Bu yetenek primer tümörü kontrol etmek için bağışıklık sisteminin emsal etmemesine rağmen belirgindir ve kanser hastaları ve hayvan modellerinde 5 in vivo kanıt yeterli destekleniyor. Önemli bir şekilde, bu bulgular, 6,7 (sitotoksisite deneyleri ile ölçülen) çoğu zaman, in vitro ve en otolog tümör hücreleri, insan ve hayvanların kan örneklerinde lökosit dolaşımı sitotoksisite dirençli olduğunu rapor ex vivo çalışmalar, tutarsız. Bu çelişki gibi afore gibi farklı lökosit popülasyonlarının, in vivo varlığı bağlanabilirbahsedilen MP lökosit popülasyonu ve aktive NK hücreleri 3 özel olarak alt popülasyonu. Gerçekten de, dolaşımdaki lökositlerin ve dalak dirençli olduğu bulunmuştur sinjenik tümör hücreleri (MADB106), MP-NK hücrelerinin 3,8 ile lize edilmesi gösterilmiştir. Bu nedenle, fischer344 (F344) farelerin akciğerlerinde metastaz sözde &quot;NK dayanıklı &#39;MADB106 hücreler değil, döner ya da genel olarak erişim kolaylığı verilen incelenen olan dalak NK hücreleri, MP-NK hücreleri tarafından kontrol edilir. Saflaştırılmış ve aktif MP-lökosit akciğer doku taşlama veya biyolojik bozulma 9 dayalı akciğerler, lökositlerin standart hasat yöntemleri ile erişilemez. Bizim yaklaşımımız doku işleme yaklaşımları ile karşılaştırıldığında iki önemli avantaja sahiptir. İlk olarak, perfüzyon yaklaşımı seçici diğer akciğer parankim, geçiş reklamı kaynaklanan hücreler ve bronş-alveol karş onları ayıran, MP-lökositleri hasadındepartmanlarında. İkincisi, daha iyi perfüzyon tekniği öğütme ve biyolojik işleme aksine bütünlük ve MP-lökositlerin fizyolojik ortam, korur hasar hücreleri, kendi morfolojisi değiştirebilir ve üretim ve bağışıklık aktivitesini modüle ve özellikle NK bastıran çeşitli faktörlerin salınımını neden olduğunu yaklaşımlar sitotoksisite 10. Akciğerler kanser metastazı ve çeşitli bulaşıcı hastalıklar için önemli bir hedef organ vardır. Tüm dolaşan habis hücreleri ve enfekte olmuş hücrelerin deforme ve kapiller endotel hücreleri ve ikamet lökositlerin ile etkileşim için gereken akciğer kılcal damarlar yoluyla geçer. Bu koşullar altında, dolaşımdaki hücreleri kolayca yerleşik MP-lökositler tarafından hedeflenebilir. Bu nedenle bu bağışıklık bölmeye aktive lökositler var biyolojik avantajlı görünüyor ve farklı biyolojik, deneysel ve klinik ortamlarda bu eşsiz MP-nüfus incelemek için önemlidir. O sy dikkat layıkÇeşitli biyolojik cevap-değiştiricilerle stemic immün aktivasyonu (örneğin, poli-inozinik-polisitidilik asit (poli I: C) ya da tip-Cı CpG oligodeoksinükleotitler (CpG-Cı ODN)), dolaşımdaki lökositlerin 3 den MP-lökositler fazla aktif olduğu gösterilmiştir 8,11.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

MP-bölmesi sergi, farklı bir lökosit alt kümesi kompozisyon lökosit dolaşan karşılaştırıldı. flow sitometri analizi kullanarak, lökosit subpopülasyonunun tespit ve hem dolaşan ve MP lökositlerin kompozisyonu karakterize etmek için ölçüldü. Granülosit ve lenfositler ileri ve yan scatter göre tespit edilmiştir. Lenfositler içinde NKRP-1 parlak hücrelerin NK hücreleri, monositler gibi T hücreleri gibi, CD3 +, H1 + ve CD...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating pulmoner lökosit seçici Hasat

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K Görüntüleme.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Video: Selective Harvesting of Marginating-pulmonary Leukocytes

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Polianhidrid Nanopartiküller Bağlı Mürin Alveoler Makrofajlar Hasat ve Değerlendirme Hücresel Etkinleştirme

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Biyomühendislik

Isolation of Mouse Lung Dendritic Cells

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 ...

İmmünoloji ve Enfeksiyon

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatik lökosit seçici Hasat

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

Swan-Ganz kateter sağ kalp kateterizasyonu sonra balon ipuçlarını endotel hücre hasat

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Tıp

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Murin Akciğerinin Doğuştan Gelen ve Adaptif İmmün Hücrelerinin Tanımlanması için Akım Sitometrik Analizi

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>Akış Sitometrisi için Fare Akciğerlerinden İmmün ve İmmün Olmayan Hücrelerin İzolasyonu

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>Fare Akciğerinden Canlı Miyeloid ve Epitel Hücre Popülasyonlarının İzolasyonu

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

Fare Akciğerinden Canlı Miyeloid ve Epitel Hücre Popülasyonlarının İzolasyonu

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Manyetik Hücre Seçimi Kullanılarak İn Vitro LLEC Kültürlerinin Oluşturulması

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Yazar Gündemi: Leptomeningeal Lenfatik Endotel Hücrelerinin Hücresel Fonksiyonlarının ve Potansiyel Klinik Etkilerinin Ortaya Çıkarılması 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal Lenfatik Endotel Hücrelerinin Hasadı ve Primer Kültürü

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Marginating pulmoner lökosit seçici Hasat

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Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Marginating-hepatik lökosit seçici Hasat

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Swan-Ganz kateter sağ kalp kateterizasyonu sonra balon ipuçlarını endotel hücre hasat

Murin Akciğerinin Doğuştan Gelen ve Adaptif İmmün Hücrelerinin Tanımlanması için Akım Sitometrik Analizi

Fare Akciğerinden Canlı Miyeloid ve Epitel Hücre Popülasyonlarının İzolasyonu

Fare Akciğerinden Canlı Miyeloid ve Epitel Hücre Popülasyonlarının İzolasyonu

Fare Akciğerinden Canlı Miyeloid ve Epitel Hücre Popülasyonlarının İzolasyonu

Leptomeningeal Lenfatik Endotel Hücrelerinin Hasadı ve Primer Kültürü