Bu çalışma bir UNCF-Merck Lisansüstü Bilim Araştırma Tezi Bursu (JLM), Ulusal Kalp, Akciğer ve Kan Enstitüsü hibe RO1-HL-62.228 (JSJ) ve RO1-HL-073.990 (JSJ) tarafından desteklenmiştir.

1. Hanks &#39;Balance Tuz Çözeltisi Hazırlama (HBSS) <ol><li> Otoklav 1 L cam beher, 1000 mL dereceli silindir, ve bar karıştırın. </li><li> Çift distile su ile 900 mL işaretine otoklavlanmış 1 L cam beher HBSS toz çözüm sulandırın. </li><li> HBSS çözüm 3.4 g HEPES Na + tuz ile karıştırın ve 7,35-7,41 arasında bir değer ya da konsantre HCL veya NaOH kullanılarak pH ayarlamak. </li><li> Istenilen pH elde ve sabit kalır sonra 5 g, albümin, tamamen eriyene kadar oda sıcaklığında çözüm sığır albümin serum ekleyin. </li><li> Toplam hacmi 1000 ml işaretine kadar çözüm antimyotic, antibiyotik ve çift distile su 10 ml ekleyin. </li><li> Çözümü iyice karıştırın. </li><li> HBSS çözüm Filtre ve daha sonra kullanmak için 50 ml Falcon tüp içine ayrı. Ek tüpler dondurulmuş ve sonraki bir tarihte kullanılabilir. </li></ol> 2. Hayvan preparatlarn - Trakeal Tüp yerleştirilmesi <ol><li> (Yani forseps, makas, trakeal tüp, 3-0 siyah örgülü sütür, küçük hayvan vantilatör vb) bir trakeal tüp yerleştirilmesi için gerekli aletler ve malzeme ile bir tepsi hazırlayın. </li><li> Sıçan oranı 1 ksilizin 2 ketamin ketamin / xylazine bir kokteyl ile tamamen uyuşturulduktan sonra, sıçan, karın, göğüs ve boğaz tıraş. Derin bir anestezi devleti elde edildiğine emin olmak için, farenin ayak tutam ve ekstremitenin pedal çekilmesi için kontrol edin. Sıçan bütün kesiler yapmadan önce derin anestezi olduğundan emin olun. </li><li> 30 ° açıyla eğimli bir kesme tahtası üzerinde kaldırdı başını sıçan yerleştirin </li><li> Orta-karın alt çene altına bir cilt insizyonu olun. Trakea ortaya çıkarmak için boynun ön üçgen kasları parçalara ayır. </li><li> Trakea altında 3-0 siyah örgülü sütür yerleştirin. </li><li> Trakea kıkırdak halkaları arasında bir kesi olun.d trakeal tüp yerleştirin. Trakeal tüp trakea güvenli dikiş sıkıca bağlayın. </li><li> Küçük bir hayvan ventilatöre trakeal tüp bağlayın. </li></ol> 3. Bağışıklık Hücre İzolasyon Aracı Hazırlama <ol><li> Bağışıklık hücre izolasyon işlemi için araçların (forseps, makas, HBSS, iğne hızlı cath üzerinde yumuşak teflon 24 gauge Baxter intravasküler kesilmiş) ile bir tepsi hazırlayın </li><li> İki 10cc şırınga hazırlayın, HBSS içeren hücreleri aspire perikardiyal kese ve yedek diğeri içine tampon tanıtmak için bir şırınga HBSS doldurun. 3.2.1. Izolasyon prosedürü için orijinal uzunluğunun 1 / 2 3 / 4 iğne hızlı cath (1.6 cm) üzerinden 24 gauge Baxter intravasküler teflon ucu azaltın. </li><li> Tüm şırıngalar, tamponlar ve boş bir 15 ml Falcon Tüp (prosedür tamamlandıktan sonra çıkarılan sıvı tutmak için) buz üzerinde yerleştirin. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Bağışıklık Hücre İzolasyonu <ol><li> Kılıç şeklinde bir süreç ulaşana kadar orta hat karın duvarı linea alba boyunca bir kesi yapmak. </li><li> Biraz yanal kılıç şeklinde bir süreç başlayarak, aksilla doğru bir açıyla devam eden göğüs kafesi ile ikili kesiler yapmak. Akciğerler, kalp, perikard ya da perfore için dikkatli olun. </li><li> Sternumun her iki tarafta insizyon tamamlandıktan sonra, mediale yanal kesme, alt kaburga diyafram uzak teşrih. Not: perikard diyafram ve zarar görmemesi için sternum bağlanır hiç farkında olun. Orak şeklinde ligaman perikard karşı tarafında diyaframın bağlı olduğu notu için bir dönüm noktası olarak kullanın. Ayrıca, perikard kesilir, hücreleri izole etme yeteneğini kaybetti. </li><li> , Göğüs boşluğu ve kalp (hala perikard kapsüllenir) maruz göğüs kafesi bir tarafı hafifçe kaldırın.</li><li> HBSS şırınga alın ve sternum ve kalp (tercihen daha fazla rostral kaudal daha) arasında yaklaşık yarım perikard kesesi içinde bir nokta bulmak ve teflon ucu takın perikard her iki katmanları ile hızlı cath kısaltılabilir. </li><li> Kateter yerleştirildikten sonra, 2 ila 3 ml HBBS tampon ile perikard boşluğu doldurmak hafifçe geçin. Perikardiyal alanı genişleyen gördüğünüzde kateter yerleştirilir hemen bilecektir. Dolum tanık ise, çekilme ve hızlı cath yeniden konumlandırmak. </li><li> Perikard kesesi dolu olduğunda, diğer 10 cc şırınga ve perikard ucu yerleştirin ve tampon aspire başlar. Aynı giriş deliği kullanılabilir ya da başka bir oluşturulabilir. Özellikle kalbin alt sağlarken, emme ucu perikardiyal kese duvarları çok yakın gelmiyor emin olun. Bu perikardiyal kese ve hücre içeren hücre kaybı delinmesiyle yol açabilirs </li><li> Tampon aspire ederken, kateter ucu mümkün olduğunca tampon toplamak için perikardiyal kese içinde dolaştı olabilir. </li></ol> Not: Bunu yaparken biraz perikard yırtılma ve orijinal bir delik daha büyük kalp üstündeki delik, panik yapmayın . Perikard sürece hala yeterince kalp çevreleyen tampon daha az miktarda da olsa kalp üzerinde hala başka bir yıkama gerçekleştirebilirsiniz. Optimal miktarda hücre toplama, 03:57 ek yıkar ihtiyaç vardır. <ol start="9"><li> Ayıklanan tampon buz üzerine yerleştirildi Falcon tüpler içine yerleştirin. </li><li> Kalp, akciğerler ve / veya daha ileri çalışmalar için gerekli olan diğer doku varsa çıkarın. Bağışıklık hücreleri ve diğer dokulara toplandıktan sonra, sıçan, hızlı ve insanca ötenazi olmalıdır. </li><li> 4 200g az 10 dakika ° C için toplanan bağışıklık hücre örnekleri Santrifüj Tüpler her zaman buz üzerinde tutmakKullanılmadığı zaman. </li><li> Hyclone Tampon veya HBSS 1 mL supernatant ve sulandırmak pelet çıkarın. </li></ol> Not: bağışıklık hücre izolasyon prosedürü sonra her kalp elde edilen bağışıklık hücrelerinin sayısını çok önemlidir. 5. Temsilcisi Sonuçlar: Bu toluidin mavi ve safranin O / alcian mavi ve vitray tekniği ile elde edilen izole kardiyak mast hücreleri bir örnek, 2A ve 2B rakamlar sırasıyla sunulmuştur. Şekil 3, kalp direk görülebileceği gibi, Morgan ve ark açıklandığı gibi bileşik 48/80 ve P maddesi olarak bilinen aktivatörleri ile tedaviden sonra histamin salınımı yeteneğini sürdürdü. CD4 flow sitometri 9 Temsilcisi dağılım araziler + hücreleri ve sırasıyla Şekil 4 ve 5, CD8 + hücreleri sunulmaktadır. T-lenfositler daha da tanımlanır ve bu tekniğin bir hücre popülasyonu olarak phenotyped. Kalp bağışıklık hücre izolasyon prosedürü Şekil 1. Akış şeması. <img alt="Şekil 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> Şekil 2 kalp mast hücrelerinin histolojik temsilcisi görüntüleri aşağıdaki gibi lekeli: (A) (B) toluidin mavi ve safranin O / alcian mavi. <img alt="Şekil 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Şekil 3 epikardiyal miyokard bölgeden izole edilmiş ve bileşik 48/80 (10μg/mL) ve P maddesi (10μM) ile uyarılan kalp mast hücreleri fonksiyonel çalışma. <img alt="Şekil 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Şekil 4 izole CD4 + hücreler, P maddesi (1:100 dilut ile etiketlenen Temsilcisi flow sitometri dağılım arazileriyon). <img alt="Şekil 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Şekil 5, P maddesi (1:100 dilüsyon) ile etiketlenmiş izole CD8 + hücreleri Temsilcisi flow sitometri .

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. 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Biz ifşa etmek başka bir şey var.

Kardiyak bağışıklık hücrelerini izole Bu yöntem genellikle enzimatik sindirimi geleneksel bir izolasyon tekniği sırasında ortaya çıkan istenmeyen aktivasyon olmadan bunların fonksiyonları, fenotip ve olgunluk analizi sağlar. Bu tekniği ile elde edilen hücrelerin ortalama toplam sayısı sıçan kalbinde başına 400.000 civarındadır ve bu hücrelerin diferansiyel boyama ve flow sitometri analizi, T-lenfositler (~% 70), makrofajlar (~% 12) ve mast hücreleri ile karışık bir nüfus olduğunu gösteri...

<table border="1"><tbody><tr><td> Reaktif ve Ekipman Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> Tom Hanks &#39;Dengeli Tuz </td><td> Sigma </td><td> H4891 </td><td> Bir şişe Hanks &#39;Denge Tuz 1 L yapar Çözümü. Sulandırın Hanks &#39;Denge Tuz Çözüm prosedürünün bir parçası olarak. </td></tr><tr><td> Antibiyotik-Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> Hepes </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Sığır Serum Albumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 ml Nalogene Filtre Şişe </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCl </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> 6 N HCL eşit hacimlerde eklemek, deiyonizesu. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> 5 N için eşit miktarda NaOH, deiyonize su ekleyin. </td></tr><tr><td> Çift Distile Su </td><td></td><td></td><td></td></tr></tbody></table> Özel Ekipman Tablo: <table border="1"><tbody><tr><td> Reaktif ve Ekipman Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> 3-O Sütur İpek Örgülü </td><td> Ethicon Ürünler </td><td> SA64H </td><td></td></tr><tr><td> Küçük Hayvan Vantilatör </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Solunum hızı dakikada 40,5 nefes ayarlayın. </td></tr><tr><td> Konik 18 iğne </td><td> BD Biosciences </td><td> 305199 </td><td> Trakeal tüp olarak kullanmak için kendi orijinal boyunun 1 / 2 3 / 4 iğne kesin. </td></tr><tr><td> 24-gauge Baxter hızlı cath kateter Teflon İpucu (iğne kaldırıldı) </td><td> Baxter </td><td> 2N111 </td><td> 1 / 2 3 / 4 Teflon İpucu süresini azaltın. </td></tr><tr><td> 10 cc şırınga </td><td> Fisher Scientific </td><td> 14-823-16B </td><td></td></tr><tr><td> 15 ml Falcon Tüpleri </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B Soğutmalı Santrifüj </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Ön-cool unutmayın ve 4 ° C </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemasitometre </td><td> Hausser Bilimsel </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Kardiyak bağışıklık hücreleri, birçok kalp hastalıkları patolojik remodeling oynadıkları roller için faiz kazanıyor mast hücreleri, T hücreleri ve makrofajlar 1-5 Bu bağışıklık hücreleri, depo ve sitokinler ve dahil olmak üzere çeşitli biyolojik olarak aktif mediatörlerin serbest triptaz olarak proteazlar 6-8 Bu aracılar, ekstraselüler matriks metabolizmasında anahtar oyuncular matriks metalloproteinazların aktive veya modüle kardiyak fibroblastlar &#39;fonksiyonu kollajen birikimi neden olduğu gösterilmiştir. 9-11 Bununla birlikte, kardiyak bağışıklık hücrelerini izole etmek için uygun teknikler var miyokard doku sindirimi bakteriyel kollajenaz kullanımı nedeniyle sorunlu olmuştur. Bu teknik, bağışıklık hücrelerinin aktivasyonu ve böylece fonksiyon kaybına neden olur. Örneğin, kalp mast hücrelerinin degranülasyonu neden olan bileşiklerin önemli ölçüde daha az duyarlı hale gelir. 12 Bu nedenle, biz t sağlayan bir teknik geliştirdikalp hastalığı bu hücrelerin rolü daha iyi anlaşılmasına yol açacak fonksiyonel kardiyak bağışıklık hücrelerinin izolasyonu 13, 14 Bu yöntem, kalbin anatomik sıçan kılıç şeklinde süreci, koltuk altı ve orak şeklinde ligaman konumu ve perikard ile bir aşinalık gerektirir. Bu simge prosedürün başarısını artırmak ve kalp bağışıklık hücrelerini daha yüksek bir verim sağlamak için önemlidir. Bu izole kardiyak bağışıklık hücrelerini sonra, fenotip, olgunluk, ve diğer kalp hücreleri ile ko-kültür deneyler, bunların karşılıklı etkileşimi daha iyi bir anlayış kazanmak için karakterizasyonu için kullanılabilir.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Bu yöntem kalp fonksiyonel bağışıklık hücrelerini izole etmek için, bu hücrelerin azalmış yanıt ile sonuçlanan istenmeyen bağışıklık hücre aktivasyonuna neden olur kollajenaz sindirim, geleneksel yöntemlere alternatif sunuyor. Izolasyon yöntemi, enzimatik sindirim ile ilgili sorunlar kaçınarak fonksiyonel kardiyak bağışıklık hücreleri elde edilir.

Fonksiyonel Kardiyak Bağışıklık Hücreleri İzolasyon

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

15.0K Görüntüleme.  University of South Carolina- School of Medicine. Bu yöntem kalp fonksiyonel bağışıklık hücrelerini izole etmek için, bu hücrelerin azalmış yanıt ile sonuçlanan istenmeyen bağışıklık hücre aktivasyonuna neden olur kollajenaz sindirim, geleneksel yöntemlere alternatif sunuyor. Izolasyon yöntemi, enzimatik sindirim ile ilgili sorunlar kaçınarak fonksiyonel kardiyak bağışıklık hücreleri elde edilir.

Video: Isolation of Functional Cardiac Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Fonksiyonel ve Moleküler Çalışmalar Birincil murin Tip II alveol epitel hücrelerinde akım sitometri İzolasyonu

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Yağ Doku Bağışıklık Hücre İzolasyonu

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periyodik Asit Schiff Boyama ile periferik kan mononükleer hücreleri glikojen algılama

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

İzolasyon, Karakterizasyonu ve Dişeti Bağışıklık Hücre Ağı Fonksiyonel Sınav

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Yalıtım ve nötrofil kaynaklı Microparticles karakterizasyonu için fonksiyonel çalışmalar

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...