Yazarlar teknik yardım için Bayan Liana Mackiewicz teşekkür etmek istiyorum. Bu eser Victoria Eyalet Hükümeti Operasyonel Altyapısını Destekleme ve Avustralya Hükümeti Ulusal Sağlık ve Tıbbi Araştırma Konseyi IRIISS ve Proje Hibe 1031212 sayesinde mümkün oldu.

1. P. ile Fare enfeksiyonu berghei-ANKA <ol><li> Defrost kriyoprezerve P. bir kısım berghei ANKA pRBC. </li><li> Iki elli emniyet tekniği kullanılarak serebral sıtma dirençli BALB / c donör fare (8-12 hafta-eski) dizginlemek. Bir 1 ml insülin şırınga (28G iğne) kullanarak pRBC 100-200 ul fare enjekte. Rutin olarak, 1-2 verici farelere enjekte edilir. </li><li> Enfeksiyon sonrası 4-5 gün hakkında (pi) kafes donör fare çıkarın ve bir iş istasyonu veya bir tek çalışma yüzeyi üzerine yerleştirin. </li><li> Yavaşça kuyruk sonuna fare dizginlemek ve küçük makas kullanarak (yaklaşık 1 mm) kuyruk ucu kesilir. Alternatif olarak, 25 G iğne kullanılarak küçük bir kuyruk ponksiyon olabilir. </li><li> Bir buzlu uç mikroskop lamı sonuna farenin kuyruk damarından kan bir damla yerleştirin. </li><li> 45 ° açı ile ilgili ikinci bir slayt (yayıcı) yerleştirin ve kan damlasını içine geri. Kan kenarı boyunca yayılır sonra, serpme-slayt arifesinde itinkan yayması yapmak için mikroskop lamı üzerinde nly. Smear kurumaya bırakın. </li><li> 10 dakika için taze hazırlanan Giemsa slaytlar leke sonra 30 sn için% 100 metanol kan smear Fix. </li><li> 1 dakika boyunca su ile çalışan yaymada durulayın, hava-kuru izin ve mikroskop (100x, immersiyon yağı) altında slayt inceleyin. 1000 eritrositler içinde pRBC numaralandırmak ve yüzde parazitemi hesaplayabilirsiniz. Onlar deney hayvanı enfeksiyon için bled önce Donör farelerin 2.5-5% arası parazitemi düzeylerine ulaşmak gerekir. </li><li> Bir heparinize mikro-hematokrit kapiller tüp kullanarak retroorbital kanama ile donör fare kan toplayın. Tüm deneyler Walter &amp; Eliza Hall Institute Hayvan Etik Komitesi gereksinimleri ile uygun olarak gerçekleştirilir. Yerel Hayvan Etik Kurulu gereksinimlerine bağlı olarak diğer kanama prosedürler kullanılabilir. 1X10 pRBC/0.2 6 ml &#39;lik bir nihai konsantrasyon RPMI medyumu içinde kan gerekli miktarda eritilir. DüşününNormal bir fare hematokrit ~ 6x10 9 kırmızı kan hücrelerinin / 1 ml &#39;dir. </li><li> Intraperitoneal (ip) 1x10 6 pRBC/0.2 ml enjekte edilerek deneysel C57BL / 6 fareler (8-12 haftalık) Infect. </li><li> Enfekte farelerde parazitemi düzeylerini izlemek için adımları 1.3-1.8 izleyin. Parazitemi günde 2 ya da 3 pi başlayarak 2-3 gün her bir tespit edilmesi gerekmektedir </li><li> Fareler kambur görünümü olarak tezahür olabilir C57BL içinde / 6 şiddetli sıtma Onset, kürk ve düşük aktiviteli ruffed. Bazı fareler bu aşamada kurtarmak olabilir, ama kendini doğrulma refleksini kaybı ilerleme geri dönüşümsüz hastalık gösterir ve hayvan ötenazi gerekir. </li></ol> 2. Fareler ve Beyin Extraction Intracardial Perfüzyon <ol><li> Bir 60 cm kolon stand üst bağlı bir kolon için bir tutucu 500 ml rezervuar sabitleyerek bir kılavuz ağırlık perfüzyon sistemi birleştirin. Rezervuarın alt ucuna plastik boru bağlayın ve tampon akışını kontrol etmek için bir boru kelepçesi sabitleyin. Bir 23 G iğne hortumu takın. FilPBS (22 ° C&#39;de tutulur) ile rezervuar l, kelepçe ve tüm hava kabarcıklarını çıkarmak için tüp aracılığıyla çalıştırmak için tampon izin açın. </li><li> Ötenazi P. CO 2 Solunması berghei ANKA-enfekte fare. Pedalı, orbital ve solunum tepkilerin yokluğu ölüm onaylayın. </li><li> Dorsal bir plastik tepsi içinde bulunan bir styrofoam diseksiyon gemide arka ve ön pençeleri tarafından ötenazi fare Pin. Yerel Hayvan Etik Komitesi gereksinimleri anestezi bağlı intracardial pefusion önceden başlaması idare edilebileceği. </li><li> % 70 etanol ile ventral tarafta silin. </li><li> Göğüs boşluğu göstermek için orta hat boyunca cildi açmak için büyük bir makas ve forseps kullanın. Yanlarına Kıvrım ve pin cilt. </li><li> Ince forseps ile sternumun tutun ve diyafram kesmek ve sternumun her iki yanında kaburga severing. Herhangi bir büyük kan damarlarına zarar vermemeye özen gösterin. </li><li> Kafasına gevşek yanındaki sternum ile göğüs kafesi Pin. </li><li> Ventriküller zekâ tutunh ince forseps ve ince makas ile dikkatlice kazımak sağ atrium. </li><li> PBS çalışırken asendan aorta doğru sol ventrikül içine yerçekimi perfüzyon sistemine takılı 23G iğne takın. Iğne ucu sadece 0.5 cm yerleştirin. </li><li> 5 dakika için ya da atık kadar fare serpmek açıktır. </li><li> Ayır farenin ve karın üzerinde yatıyordu. % 70 etanol ile kafasını temizleyin. </li><li> Büyük makas kullanarak, sadece servikal spinal kord alanı üzerinde bir kesim yapmak. </li><li> Medyan kaudal-rostral kesim kafatası maruz uzak kafatası tabanında başlayan ve cilt soyma yapmak için ince makas kullanın. </li><li> Başımı tutarak, her yörünge boşluğuna küçük bir makas bıçakları yerleştirin ve yörüngeleri arasında bir kesim yapmak. </li><li> Sutur boyunca uzunlamasına kesilmiş olun ve dikkatlice dışarı doğru her beyin yarımkürede kafatası kabuğu. </li><li> Bir spatula kullanarak, yavaşça beyin kaldırın ve RPMI medyumu içeren 10 ml santrifüj tüpüne yerleştirin. </li></ol>3. Beyin-sekestre Lökosit izolasyonu (BSL) <ol><li> Bir güvenlik kabini içinde çalışarak, yerde taze RPMI doku kültür ortamı 3-5 ml içeren bir Petri kabındaki bir paslanmaz çelik hücre süzgecinden (40-60 mesh boyutu) üstüne beyin hasat. </li><li> Küçük parçalar halinde beyin dokusu kesin. </li><li> Bir kristal piston kullanarak hücre süzgecinden beyin dokusunun küçük parçalar bastırın. </li><li> 4, 10 dakika boyunca 250 ° C&#39;de xg&#39;de 10 ml tüp ve santrifüj ile beyin Homojenat, transfer </li><li> % 0.05 collagenase, D ve 2U/ml DNAz I içeren RPMI medyumu, 3 ml içinde çözülür pelet </li><li> Oda sıcaklığında 30 dakika için karışım döndürün. Bir 70 mikron naylon hücre süzgecinden ve / ya da itme karışım 5 dakika süreyle buz üzerinde inkübe ederek hücre artıkları çıkarın. </li><li> Oda sıcaklığında 20 dakika için 400 x g&#39;da (herhangi bir fren) bir 7 ml% 30 Percoll yastık ve santrifüj üzerine beyin Homojenat yerleştirin. </li><li> 5 dakika için buz üzerinde kırmızı kan hücresi lizis tamponu ile inkübe 1 ml pelet yeniden süspanseintracardial perfüzyon beyin kaldırılır olamazdı yapışık pRBC, lyse. </li><li> RPMI medyumu 9 mL, 4 ° C&#39;de 10 dakika boyunca 250 x g santrifüj ile hücreler yıkamak </li><li> RPMI medyumu ile bir Eppendorf tüpü transfer hücre 50-100 ul içinde süspanse BSL içeren pelet. </li><li> Yaşayabilir hücrelerin tanımlanması için Tripan mavi hücre örneği bir 5-10 ul alikot 1:02 seyreltin. </li><li> Hemasitometre kullanarak mikroskop altında canlı hücreleri saymak. Günde 6 pi itibaren, P. Gönderen berghei ANKA-enfekte farelerde 20,000-100,000 BSLs telafi edilebilir, nörolojik belirtiler gösterir. </li></ol> 4. Multicolour akış sitometri yöntemi ile BSL immünofeotipleme <ol><li> 4, 10 dakika boyunca 250 ° C&#39;de x g&#39;de santrifüjleyin hücreleri, (PBS,% 1 dana fetus serumu (FCS), 2 mM EDTA) ile boyanarak 50 ul tampon içinde tekrar süspansiyon topak süpernatantı ve aspire. </li><li> Anti-CD16/32 antikor (Fcblock) saflaştırılmış 1 ul ekle. </li><li> 1 için hücreler inkübeBuz üzerinde 0 dk. </li><li> Boyama Tampon 1 ml ile hücreleri yıkayın. 4 ° C &#39;de 10 dakika boyunca 250 x g&#39;de santrifüjleyin hücreleri ve süpernatan aspire. </li><li> Gerekli floresan antikor önceden belirlenmiş en uygun seyreltmeler, yani anti-CD4, anti-CD8, anti-TCR ve anti-NK1.1 ihtiva eden tampon Boyama 50 ul içinde süspanse hücreler. </li><li> Buz üzerinde 1 saat boyunca inkübe edin. </li><li> Boyama Tampon 1 ml ile hücreleri yıkayın. 4 ° C &#39;de 10 dakika boyunca 250 x g&#39;de santrifüjleyin hücreleri ve süpernatan aspire. </li><li> PBS içinde 100 ul hücre içinde yeniden süspanse edin. </li><li> Bir akım sitometri plastik tüp hücreleri aktarın. </li><li> Bir akış sitometresinin kullanarak, en az 5.000-10.000 olaylar kazanmak. Gerektiği gibi uygun boyanmamış hücre kontrolleri ve florokrom tazminat örnekleri dahil edilmelidir. </li><li> Uygun akım sitometri yazılımını kullanarak sonuçları analiz edin. </li></ol>

<ol>
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Çıkar çatışması ilan etti.

BSL izolasyonu ve karakterize edilmesi ve analiz deneysel fare modellerinde doku hasarı ya da enfeksiyona yanıt olarak beyin göç inflamatuar hücrelerin miktarının sağlayan bir yöntem. Organ ekstraksiyonu ve bunu izleyen hücre izolasyonu önce kan bölmesinin çıkarılması için bir intracardial perfüzyon adım giriş non-inflamatuar dolaşımdaki lökositler ile inflamatuar hücrelerin kirlenmesini önlemek için yararlıdır. Bu inflamatuar hücreler beyin parankimi 5 nüfuz hangi tür kemirgen ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td></td><td></td><td></td><td> Çözümler ve tamponlar </td></tr><tr><td> Giemsa en azur eosin metilen mavisi çözeltisi </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Distile su 1:10 seyreltme </td></tr><tr><td> RPMI medyumu </td><td></td><td></td><td> Fare tonisite </td></tr><tr><td> Fare tonisite PBS </td><td></td><td></td><td> 20 mM sodyum fosfat, 0.149 NaCl, pH 7.3 </td></tr><tr><td> % 0,4 Tripan Mavisi </td><td> Sigma Aldrich </td><td> T-8154 </td><td> 1:02 dilüsyon </td></tr><tr><td> Kollajenaz D </td><td> Biyokimyasal Worthington </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAse) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> Sığır pankreas Gönderen </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> PBS içinde% 30 çözelti </td></tr><tr><td> Ultra Saf Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Amonyum Klorür (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Kırmızı Hücre Lizis Tampon </td><td></td><td></td><td> 17 mM Tris, 14nm NH4CI, pH 7.2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA disodyum tuzu </td><td> Merck </td><td> 10093.5V </td><td> 0.1M, pH 7.2 </td></tr><tr><td></td><td></td><td></td><td> Antikorlar ve konjugatlarının </td></tr><tr><td> Anti-fare CD16/CD32 (Fc Blok), klon 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553.142 </td><td> 50 ul boyama tampon içinde 1 ul (0.5mg/50 ml) </td></tr><tr><td> FITC-anti-fare CD4, klon H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-fare NK1.1, klon PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-anti-fare CD8, klon 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-fare anti-TCR-β, klon H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-fare CXCR3, klon 220.803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinlenmiş-anti-fare CCR5, klon C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Steptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman ve malzeme </td></tr><tr><td> SuperFrost mikroskop lamı </td><td> Lomb Menzel-Glaser </td><td></td><td></td></tr><tr><td> Diseksiyon forseps, makas </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml PBS rezervuar </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Kauçuk hortum </td><td></td><td></td><td></td></tr><tr><td> 23G iğne </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Hücre ayrışma kiti metal elek içeren </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 mikron naylon hücre süzgecinden </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Hemasitometre </td><td> GmbH Neubauer </td><td> 717810 </td><td></td></tr><tr><td> Flow sitometri tüpler </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Biz P. beyin kan damarlarından yapışık inflamatuar hücrelerin izolasyonu ve karakterizasyonu için bir yöntem tarif berghei ANKA-enfekte fareler. Deneysel serebral sıtma indüksiyon, bir nörolojik sendromu bu parazitin suşu sonuçlar duyarlı fare suşlarının enfeksiyonu insanlarda 1,2 Plasmodium falciparum aracılı şiddetli sıtma özetlediği bazı önemli yönleri olduğunu. Kan aşama sıtma Olgun formlar vasküler endotel hücreleri ile bağlamak sağlar enfekte eritrosit, yüzeyi üzerinde parazit proteinleri ifade eder. Bu süreç hipoksi ve kanamalar 3 sonuçlanan kan akımında engel indükler ve aynı zamanda parazit sekestrasyon sitesine inflamatuvar lökositlerin işe uyarır. Diğer enfeksiyonlar, örneğin neutrotopic virüsler 4-6, sıtma-parazitlenmiş kırmızı kan hücreleri (pRBC) yanı sıra ilişkili yatkın aksine, hemekspirayon lökositlerin kan damarları içindeki yerine beyin parankimi infiltre daha münzevi kalır. Böylece non-inflamatuar dolaşan hücre, organ çıkarma ve doku işleme öncesi enfekte-farelerin geniş intracardial perfüzyon ile sekestre lökositlerin kirlenmesini önlemek için kan bölmesi çıkarmak için bu prosedürü gereklidir. Perfüzyon sonra, beyinleri hasat edilir ve küçük parçalar halinde disseke. Dokuların yapısı daha da oluşan beyin homojenat sonra miyelin ve diğer doku enkaz beyin sekestre lökositlerin ayrılması (BSL) sağlayan bir Percoll degrade üzerinde santrifüj edilir Kollajenaz D ve DNAz I. ile enzimatik tedavi ile bozulur. İzole hücreler daha sonra yıkanır, flow sitometri ile sonraki analiz için floresan antikorlar ile hemasitometre ve lekeli kullanılarak sayıldı. Bu prosedür inflamatuvar lökositlerin kapsamlı fenotipik karakterizasyonu re beyin göç veriyorinme gibi viral veya paraziter enfeksiyonlar gibi çeşitli uyarılara yanıtı. Yöntemi de klinik öncesi hayvan modellerinde yeni anti-inflamatuvar tedaviler değerlendirilmesi için yararlı bir araç sağlar.

Şekil sonuçlanır. 2 gösteri yüzdeleri ve perfüze veya unperfused sıtma ile enfekte olmuş ve naif kontrol farelerin beyinleri kurtarıldı farklı BSL nüfus mutlak sayıları. Protokol metninde de belirtildiği gibi İzole BSL PE-anti-NK1.1 ve APC-anti-TCR-β antikorları ile boyandı. Önceki bulguları 7-9 ile uyumlu olarak, αβTCR + T hücreleri perfüze sıtma ile enfekte farelerde (günde 6 pi) ve beyinlerinde BSL havuzu yüksek oranda oluşur. Bu nüfusun önemli ölçüde (Şekil 2A...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Beyin kan damarlarının gelen yapışık inflamatuar lökositlerin izolasyonu için bir yöntem<em&gt; Plasmodium berghei</em&gt; ANKA ile enfekte olmuş fareler tarif edilmektedir. Yöntemi flow sitometri ile floresan antikorlar ve müteakip analizler ile boyanarak miktarının yanı sıra izole lökositlerin fenotipik karakterizasyonu sağlar.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

isolation-analysis-brain-sequestered-leukocytes-from-plasmodium

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

12.7K Görüntüleme.  The Walter and Eliza Hall Institute of Medical Research. Beyin kan damarlarının gelen yapışık inflamatuar lökositlerin izolasyonu için bir yöntem Plasmodium berghei ANKA ile enfekte olmuş fareler tarif edilmektedir. Yöntemi flow sitometri ile floresan antikorlar ve müteakip analizler ile boyanarak miktarının yanı sıra izole lökositlerin fenotipik karakterizasyonu sağlar.

Video: Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

Beyin-infiltre lökositler izolasyonu

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

İmmünoloji ve Enfeksiyon

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

İnsan lökosit ve trombosit İzolasyonu ve Değerlendirme Protokolleri: Biyoenerji ve Oksidatif Burst

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Akış adlı lökositlerin üzerine Stromal hücreler Etkilerinin İncelenmesi

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

İnsan Maternal-Fetal Arabirimi lökosit İzolasyonu

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Maternal Fetal Arayüz at Fare Dokular lökosit İzolasyonu

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu<em&gt; Plasmodium</em&gt; Sporozoitler

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...