Bu çalışma FL bir Amerikan Kalp Derneği Bursu Ödülü (16POST29950007), Sağlık (NIH T32 HL069765) BLD, MA Salih bir Amerikan Kalp Derneği Bursu Ödülü (14POST20420025) için National Institutes of bir eğitim hibe ve bir NIH tarafından desteklenen K08 ödülü MSM (HL121671). MSM de Gilead Sciences, Inc. bir araştırma bursu ile desteklenmektedir

Vanderbilt Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi Burada tarif edilen prosedürler onayladı. Fareler ev sahipliği ve Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu uyarınca bakılan (Ulusal Akademiler Press. Revize 2010). Fareler ile Aort lenf düğümleri, Böbrek ve Aort 1. İzolasyonu <ol><li> CO 2 inhalasyon fareler Euthanize. % 70 etanol ile göğüs püskürtün ve dikkatle deri ve kalp maruz makas ile göğüs duvarı açın. </li><li> damarsal serpmek için, sağ atrium küçük bir kesi yapmak ve sürekli bir 21 ya da 23 G iğne kullanılarak sol ventrikül apeksi içine soğuk PBS (yaklaşık 1 ml / sn) en az 10 ml enjekte edilir. Tüm organlar blanched kadar serpmek. Kalp ilk blanches. karaciğer Ağartma perfüzyon iyi performans olduğunu gösterir. </li><li> küçük forseps ve ince makas kullanarak, hafifçe, kesip ve bağırsakları dışarı çekinmide, dalak, pankreas ve karaciğer daha iyi aorta görselleştirmek için. Not: Bu adım kontaminasyonu uyarabilir gastrointestinal hasar olarak çok hassas yapılmalıdır. </li><li> Cut out ve makasla her akciğer çıkarın. Bir iğnesiz şırınga kullanılarak, fosfat tamponlu tuzlu su (PBS) ile göğüs boşluğunu durulayın. steril gazlı bez kullanılarak aşırı kan ve sıvı kaldırmak. </li><li> ince diseksiyon forseps kullanılarak abdominal aort lenf düğümleri kaldırın. kapsül yırtılmasına değil emin olun. kesme forseps her lenf nodu yüzeyi üzerinde geride kalan yağ çıkarmak. ince makasla iki böbrekleri kesip. </li><li> ince, kavisli makas ile tüm aorta (torasik ve abdominal aorta) teşrih. kalp hizasında başlayın ve dikkatle uzak yemek borusu ve aort bağlı çevreleyen perivasküler yağ tutmak için emin iliak bifurkasyona kadar aşağı omurları arasından teşrih. NOT: Aort diseksiyonu daha fazla remoÇevredeki yapıların mikroskop altında yapılabilir ettik. Özellikle, arteriyel dalları (karotis arterler, subklavyen arter, çölyak, mezenterik ve renal arterler) ve lenf düğümleri kaldırın. Bu çok inflamatuar hücreler hipertansiyon ayarında ikamet bir site olduğundan her aort etrafında perivasküler yağ tutarlı bir tabaka tutun. soğuk PBS içeren tüplere, her doku yerleştirin. </li></ol> Her bir dokudan Tek hücre süspansiyonları, 2. Üretim <ol><li> böbrekler NOT: Böbrek mekanik ayrışma artı enzimatik sindirim birleştirerek tek bir hücre süspansiyonu ayrışmış edilebilir. <ol><li> (% 5 fetal sığır serumu (FBS) ile desteklenmiş) 1640 ortamı (2 mg / ml) kollajenaz D eklenmesi ve DNase I (100 ug / ml), RPMI ile Böbrek sindirim çözeltisi hazırlayın. Böbrek örnek başına 10 ml hazırlayın. </li><li> Böbreğin sindirmek 10 ml içeren bir ayrışma tüp içine iki böbreği aktarmak için forseps kullanabiliriyon solüsyonu. </li><li> üreticinin talimatlarına göre bir yarı-otomatik bir ayıracı cihazı kullanılarak mekanik ayrılma gerçekleştirin. </li><li> mekanik ayrışma sonra cihazdan tüp ayırmak ve enzimatik sindirim gerçekleştirin. sürekli dönme altında 37 ° C &#39;de 20 dakika boyunca inkübe edin. Hemen% 5 FBS ile takviye edilmiş, soğuk RPMI 1640 ortamı eklenerek enzimatik sindirim dur. </li><li> 40 mikron süzgeç vasıtasıyla filtreleme sonra 50 ml konik tüp içine pipetleme çözüm aktarın. 1 ml şırınga pistonu bastırarak birbirinden kalan doku alay. hücreler (500 xg, 8 dakika, 4 ° C) pelet. </li><li> % 36 yoğunluk gradyan santrifüj ortamının 3 ml pelletini. 15 ml konik tüp içine süspansiyon aktarın. Yavaşça herhangi bir kesinti olmadan 3 ml% 36 hücre süspansiyonu altında% 72 yoğunluk gradyan santrifüj medya 3 ml ekleyin. Santrifüj (frensiz 1000 xg, 20 dakika, 4 ° C). &lt;/ Li&gt; <li> bağışıklık hücreleri arayüzünde yer alacaktır. hücresel enkaz içeren üstteki sarımsı bir tabaka çıkarın ve sonra soğuk PBS kullanarak 15 ml hacim getirmek. arıza iyice degrade sallayın. hücreler (300 xg, 8 dakika, 4 ° C) Spin ve% 5 FBS ile RPMI 3 ml pelletini. Tripan mavisi dışlama kullanarak hemasitometre canlı hücrelerinin sayısını. </li></ol></li><li> aort <ol><li> kolajenaz A (1 mg / ml), kolagenaz B (1 mg / ml) eklenerek aort sindirim çözeltisi hazırlayın ve DNase I (100 ug / ml), RPMI ile (% 5 FBS ile takviye edilmiş) 1640 ortamı. </li><li> ince forseps kullanılarak, aort sindirim çözeltisi, 1 ml içeren 2 ml&#39;lik bir tüp içine aort aktarın. 1 ml sindirim çözeltisi içinde ince makas ile çok küçük parçalar halinde bütün aort kıyma. buz üzerinde tutun. sürekli dönme altında 37 ° C sıcaklıkta 30 dakika boyunca inkübe edin. </li><li> Bir doku kültürü çanak çözüm aktarın ve enzyma durdurmak% 5 FBS soğuk RPMI 5 kez hacmi eklenerek tlc sindirim. </li><li> 40 mikron süzgeç vasıtasıyla filtreleme sonra 50 ml konik tüp içine pipetleme çözüm aktarın. 1 ml şırınga pistonu bastırarak tek bir hücre süspansiyonu içine ayrı kalan doku alay. süzgeci yıkayın ve hücreleri (300 xg, 8 dakika, 4 ° C) pelet. </li><li> % 5 FBS ile RPMI 10 ml ekleyerek pelet yıkayın. hücreler (300 xg, 8 dakika, 4 ° C) Spin ve% 5 FBS ile RPMI 3 ml pelletini. Tripan mavisi dışlama kullanarak hemasitometre canlı hücrelerinin sayısını. </li></ol></li><li> Aort lenf düğümleri <ol><li> bir doku kültürü kabı (60 mm x 15 mm) ve 40 um süzgeç yerleştirin. süzgeç üzerinde lenf düğümlerini yerleştirin ve 1 ml şırınga pistonu bastırarak tek bir hücre süspansiyonu içine birbirinden kızdırmak. % 5 FBS ile RPMI 2 ml süzgeç yıkayın. çanak hücreleri toplamak. </li><li> transfer15 ml&#39;lik bir Falcon tüpü içine hücre ve pelet hücreleri (300 xg, 8 dakika, 4 ° C). % 5 FBS ile RPMI 500 ul pelletini. Tripan mavisi dışlama kullanarak hemasitometre canlı hücrelerinin sayısını. </li></ol></li></ol> Akış Sitometrisi ile sitokin Algılama 3. Ex Vivo Uyarım <ol><li> % 5 FBS, temel olmayan amino asitler, 0.1 mM, 50 uM β-mersaptoetanol ve% 1 penisilin / streptomisin ile RPMI: hücre kültür ortamı hazırlayın. </li><li> Santrifüj, böbrek, aort ve lenf düğümlerinden elde edilen her hücre süspansiyonu (300 xg, 8 dakika, 4 ° C). Ml başına 1 x 10 6 hücre bir konsantrasyonda, hücre kültür ortamı, her pelletini. </li><li> Hücre kültürünün her 1 ml (Phorbol 12-miristat 13-asetat (PMA), kalsiyum iyonofor iyonomisin ihtiva eden ve Golgi inhibitörü Brefeldin A) hücre aktivasyonu kokteyli 2 ul ekleyerek hücrelerini uyarır. NOT: nihai konsantrasyonPMA 81 nM PMA ile Brefeldin A Tedavi ionomisin 1.33 uM ve 5 ug / ml ve iyonomisin sitokinler üretmek için hücreleri aktive eder: her bir bileşenin s bulunmaktadır. Sitokinler tespit edilecek hücrelerde sıkışıp gereken proteinleri salgılanır. Brefeldin A endoplazmik retikulum ve Golgi aygıtında normal salgılanan proteinlerin birikmesine yol açar. Bu nedenle, burada tarif edilen teknik, akış sitometrisi ile sitokin üreten limfositlerin tespit edilebilirliğini arttırır. </li><li> 12 oyuklu 2.000.000 hücre (aort veya böbrek) ya da 24 oyuklu (lenf düğümü) düşük yapışma plakaları Levha 1. 3 saat 37 ° C nemlendirilmiş CO2 kuluçka makinesi içinde plaka koyun. Not: kaplama hücre sayısı kontrol farelerinin lenf düğümlerinde daha düşük olabilir. Ayrıca, uyarılma zaman deneysel olarak çalışılan her hücre popülasyonu ve sitokin için belirlenmelidir. aort, böbrek veya lenf düğümlerinden izole edilmiş T hücreleri durumda, 3 ila 4 saat boyunca teşvik önerilir. Daha uzun bir uyarım WHasta sitokin salgılanmasını arttırmak ve CD3, CD4 ve CD8 gibi çeşitli T hücresi yüzey markerlerine bir başka infekte hücrelerde bunu yapmaz. (Hatta 3-4 saat stimülasyon bu belirteçlerin bazı infekte hücrelerde unutmayın.) </li></ol> 4. Yüzey Boyama <ol><li> bir polistiren FACS tüpü ve santrifüj (300 xg, 8 dakika, 4 ° C) içine hücreleri aktarın. (FACS tamponu ile iki kez (Ca2 + ve Mg + serbest PBS% 4 FBS ve 2 mM EDTA ihtiva eden) hücreler yıkanır ve arzu edilen antikor panel sayısı ve gerekli kontrol tüplerinin sayısına bağlı olarak farklı FACS tüplere eşit hücre süspansiyonu bölmek aşağıya bakınız). </li><li> Her panel için tazminat gerçekleştirmek için, her doku tipi ve antikor paneli için lekesiz kontrol tüpleri ve tek lekeli tüpler hazırlamak. Buna ek olarak, tüm ama antikorların birini ekleyerek her antikor paneli için floresans eksi bir (FMO) kontrol tüpleri hazırlar. </li><li> FACS ile 2 ml her tüp ses seviyesini ayarlamakTampon. Santrifüj hücre süspansiyonu (300 xg, 8 dakika, 4 ° C), buz üzerinde 10 dakika süre ile milyon hücre başına anti-CD16 / CD32 antikorları, 0.5 ug hücrelerin inkübe edilmesi ile yüzer ve blok Fc reseptörleri çıkarın. NOT: CD16 düşük afinite IgG Fc reseptörü III (FcR III) ve CD32 FcR II. CD16 / CD32 granülositler, mast hücreleri, monositler / makrofajlar, doğal katil hücreler, B hücreleri, dendritik hücreler ve bazı aktif, olgun T hücreleri üzerinde ifade edilir. </li><li> geçerlilik boyaması yapın: 1x PBS 1000 ul 1x PBS, santrifüj (300 xg, 8 dakika, 4 ° C) ve tekrar süspansiyon hücreleri yıkayın. hücre geçirgenliği olmayan amin reaktif boya (canlılık leke) 1 ul ekle. ışıktan korumalı 4 ° C&#39;de 15 dakika süreyle inkübe edin. </li><li> İnkübasyon sırasında, FACS tamponu uygun bir hacimde (yüzey boyama) antikor kokteyli hazırlanır. FACS tampon 1-2 ml hücreler iki kez yıkanır, santrifüj (300 xg, 8 dakika, 4 ° C) ve 100 ul her bir peletAntikor karışımı. ışıktan korumalı 4 ° C sıcaklıkta 30 dakika boyunca inkübe edin. Not: antikor sitometri her bir akış sağlamak için, bir doz titrasyon eğrisi gereksinim duyulan antikor optimal miktarını belirlemek için yararlıdır. </li><li> hücreler (300 xg, 8 dakika, 4 ° C) FACS tampon, 2 ml ile iki kez hücreleri yıkayın ve pelet. </li></ol> 5. Sabitleme, Permeabilization ve Hücre içi Boyama <ol><li> Üreticinin talimatlarını takip ederek uygun tamponlar içeren bir tespit ve permeabilization kiti ile hücre içi boyama yapın. Fix ve uygun tampon onları resuspending hücreleri geçirgenliği. ışıktan korumalı 4 ° C&#39;de 40 dakika boyunca inkübe edin. </li><li> 1x Permeabilization 1 ml ekleyin ve sabit ve permeabilize hücrelere doğrudan tampon yıkayın. Santrifüj (500 xg, 8 dakika, 4 ° C) ve süpernatant kaldırmak. 1x yıkama tamponu 2 ml pelletini. </li><li> antib karışımlarını hazırlamak1x yıkama tamponu (tüp başına genellikle 100 ul) uygun bir hacimde (hücre içi boyama için) Odies. Not: Yukarıda tarif edildiği gibi, uygun antikor konsantrasyonu panelinde, her bir antikor için tespit edilmesi gerekmektedir. Örneğin, IL-17A veya IL-17F karşı antikorlar için, biz 1:30 bir seyreltme faktörü kullanın, ancak IFNy ve T-bahis antikorları için, biz 1 bir seyreltme faktörü kullanın: 100. </li><li> Santrifüj hücreleri (500 xg, 8 dakika, 4 ° C) ve antikor karışımı, 100 ul her pelletini. ışıktan korumalı 4 ° C&#39;de 40 dakika süreyle inkübe edin. </li><li> hücreler (500 xg, 8 dakika, 4 ° C) 1 x yıkama tamponu 2 ml hücreler iki kez yıkanır ve pelet. 1x yıkama tamponu 300 ul ile pelet yeniden süspanse edin ve uygun filtreler ile bir akış sitometresi üzerinde analiz. </li><li> doku örneğinin başına toplam hücre sayısının ölçümü için sayma boncuk kullanmayın. alımı öncesinde, her tüpe boncuk sayma önerilen hacim / numara ekleyin. </li></ol> 6. Tazminat, Yolluk, Normalleştirme, ve İpuçları <ol><li> Tazminat ve yolluk <ol><li> tazminat çalıştırın lekesiz ve tek lekeli kontrol tüpleri spektral örtüşme ayarlamak için. Not: dengeleme boncuklar yerine hücre bazlı dengeleme kontrol kullanımı, bir numunede ilgi veya hücre popülasyonunun bir antijen olduğu telafi zor olacak hücreler kullanılarak bu düşük seviyelerde mevcut olduğu durumlarda gereklidir. tandem boyalar kullanarak Ayrıca notun, tandem boyalar çok-çok-ve her gün değişir çünkü her bir deney için tazminat hazırlamak için tavsiye edilir. </li><li> Yeni bir renkli deney başlatırken floresan eksi panelde tüm fluorophores için bir kontrol (FMOs) çalıştırın. NOT: FMO kontrolleri biri hariç panelde tüm antikorlar içeren örneklerdir. Bu kontroller düzgün rla ma k atlanmış antikor negatif sinyallerin karşı pozitif doğru ayrımcılığa izinKapıları t. Bu kontroller ifade seviyeleri hedef nüfusun ayrılması kötü olduğunda ya da (örneğin, zaman hedef bir sitokin veya transkripsiyon faktörüdür) düşük olduğunda özellikle önemlidir. bunlar, antijen spesifikliği olmayan antikor izotipinin ve fluorofor konjügatı için uygun telafi sağlamak gibi FMO kontrol her zaman gerekli olmasa da, bazı durumlarda, izotip kontrol yerde tercih edilebilir. </li><li> Birincil kapıyı kurmak: lökosit nüfusu tespit etmek amacıyla Forward Scatter Area (FSC-A) ve Side Scatter Area (SSC-A) Gerilimi ayarlamak. </li><li> ölü hücreleri hariç. NOT: canlılık leke yüzeyi ve hücrelerin iç hem de mevcut ücretsiz aminler ile reaksiyona girer. hücrelerin yaşama aksine, (tehlikeye diyaframlarla) ölü hücreler, protein etiketleme miktarının artırılması, boya sitoplazmaya girmesine izin verir. Böylece, ölü hücreler kolay ayrımcılık sağlayan canlı hücreler daha parlak olacaktır. Canlı üzerinde KapısıHücreler. Not: aort ve böbrek hücre süspansiyonları canlılığı nedeniyle ek sindirim aşamasından lenf düğümleri hücre süspansiyonları canlılığı daha düşük olabilir. </li><li> Arsa İleri Yayılım Alanı (FSC-A) [y-ekseni] ve İleri Yayılım Yükseklik (FSC-H) [x ekseni]. Mayoları bu nokta arsa üzerinde bir diyagonal olarak görünür. Bu diyagonal Kapısı ikilileri dışlamak için. Sonra SSC-A [y ekseni] ve CD45 [x eksenini] arsa. </li><li> CD45 + lökosit nüfus kolaylıkla tespit edilebilir. Bu nüfus Kapısı. Takip eden popülasyonlarının Bu popülasyondan tespit edilebilir. </li></ol></li><li> Hücre sayımlarının Normalleştirme <ol><li> Sayma boncuklar düşük FSC ve yüksek SSC neden özelliklere sahiptir. 50.000 boncuk tek bir hücre süspansiyonu 300 ul ilave edilir, aşağıdaki gibi tüp başına Örneğin, herhangi bir mutlak sayısının hesaplanması için denklemi hücre tipi vermek: Hücre sayısı per tüp = (akış sitometresi tarafından sayılan boncuk 50,000 / sayısı) sayılan hücrelerin sayısı x. Bu hesaplama kullanarak, tüp başına herhangi bir hücre tipinde mutlak sayısı belirlenebilir. Her organ elde kaç borular göre, organ başına hücre sayısı hesaplanabilir. Not: protokolü 4 ° C&#39;de tüm hücreleri ve reaktifler tutun. bu hücrelere zarar verebilir, çünkü kuvvetli vorteks kaçının. pelet hücreleri çok az güçleri kullanın. hücre ölümünü hızlandırabilir beri FACS tüp içinde kabarcıklar yapmaktan kaçının. topaklar, herhangi bir zamanda kurumasına izin vermeyin. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genomics.">Single-cell genomics.</a> Nat Methods. 8 (4), 311-314 (2011).</li><li>Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry:+Today's+technology+and+tomorrow's+horizons.">Cytometry: Today's technology and tomorrow's horizons.</a> Methods. 57 (3), 251-258 (2012).</li><li>Jiang, L., Tixeira, R., Caruso, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+progression+of+cell+death+and+the+disassembly+of+dying+cells+by+flow+cytometry.">Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.</a> Nat Protoc. 11 (4), 655-663 (2016).</li><li>Lemoine, S., Jaron, B., Tabka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dectin-1+activation+unlocks+IL12A+expression+and+reveals+the+TH1+potency+of+neonatal+dendritic+cells.">Dectin-1 activation unlocks IL12A expression and reveals the TH1 potency of neonatal dendritic cells.</a> J Allergy Clin Immunol. 136 (5), 1355-1368 (2015).</li><li>Gaublomme, J. T., Yosef, N., Lee, Y., 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=Single-Cell+Genomics+Unveils+Critical+Regulators+of+Th17+Cell+Pathogenicity.">Single-Cell Genomics Unveils Critical Regulators of Th17 Cell Pathogenicity.</a> Cell. 163 (6), 1400-1412 (2015).</li></ol>

MSM Gilead Sciences Kardiyovasküler bir hibe ile desteklenmektedir.

The protocol described herein has been optimized to properly identify T cell subsets present within murine kidneys, aorta and lymph nodes. This protocol can be easily adapted to examine other immune cell subsets such as B lymphocytes and innate immune cells and can be modified to include other tissue types. The digestion step is critical and has to be modified and optimized for each tissue9. A prolonged digestion step or the use of an inappropriate enzyme can affect the stability of antigen expression. Similar...

Son kanıtlar, adaptif bağışıklık sisteminin, özellikle de T lenfositler, çok sayıda kalp-damar hastalıkları 1,2,3,4,5 gelişiminde önemli bir rol oynadığını göstermektedir. Örneğin, anjiyotensin II kaynaklı hipertansiyonu modelinde, gemiler ve farelerin böbreklerinde T hücrelerinin birikimi 6 tarif edilmiştir. damar birikim adventisya ve perivasküler yağ ağırlıklı olarak. Böbrekte, T hücreleri, medulla ve renal korteksten hem de birikir. Dahil olduğu alt kümesi bağlı olarak, bu T hücreleri vasküler ve böbrek fonksiyonlarını etkileyebilir ve patolojinin gelişmesine yol açabilir farklı sitokinler doğuran (McMaster ve ark., 6 tarafından gözden). CD4 + T yardımcı lenfositler çok alt-grup halinde ayrılabilir: fonksiyonları ve imza sito göre T yardımcı 1 (Th1), Th2 Th9, Th17, Th22, regülatör T (regülatör) hücreleri ve T yardımcı foliküler (TFH) hücrelerkines 7. Benzer şekilde, CD8 + sitotoksik T hücreleri Tc1, TC2, Tc17 ya TC9 8 olarak sınıflandırılabilir. (CD4 veya CD8 T hücresi işaretlerini ifade yoktur, yani hücreleri) çift negatif T hücreleri bulunmaktadır. Bu hücrelerin bir alt alternatif bir gama delta T hücre reseptörü (yerine klasik alfa ve beta reseptörleri) sahiptirler ve bu nedenle, gama, delta T hücreleri olarak ifade edilir. yüzey işaretleyici, sitokin ve transkripsiyon faktörü flow sitometri çok parametre analizi bu hücreleri tanımlamak için en iyi yaklaşım oluşturmaktadır. Bu yöntem geniş bir immünoloji alanında kullanılmasına rağmen, bu daha az katı organlar ve kalp-damar hastalıklarının bir ortamda tarif edilmektedir. Tarihsel olarak, dokularda lenfosit tanımlama immünohistokimya ya da RT-PCR yaklaşımlar ile sınırlı kalmıştır. immünohistokimyasal ve immünofloresan int bir antijenin doku dağılımını belirlemek için güçlü yöntemler olmasına rağmenerest, onlar fenotipik ilgili alt grupları tanımlamak için yetersiz kalmaktadır. RT-PCR analizi antijenleri, sitokinler ya da transkripsiyon faktörleri mRNA ekspresyonunu tespit etmek için yararlıdır Buna ek olarak, bu aynı zamanda tek tek hücreler seviyesinde çok proteinlerin tespiti izin vermez. sitokinler ve transkripsiyon faktörleri tespit etmek için hücre içi boyama ile birlikte, özellikle flow sitometri gelişi, katı organlarda immün hücre alt tek hücre düzeyinde belirlenmesi ve niceliksel olanak sağlayan güçlü bir teknikle araştırmacılar sağlar. Bu, anjiyotensin II kaynaklı hipertansiyonu modelinde kemirgen böbrek, aort ve aort lenf düğümleri içinde bulunan ana T hücre alt akış sitometrisi ile tespit etmek, bir hücre içi boyama tahlili optimize ettik. Çok yeniden bir doku sindirim, ex vivo aktivasyonu, permeabilization ve yüzey ve hücre içi boyama sonuçları: her adımın optimizasyonuDiğer kardiyovasküler ve böbrek hastalığı modellerinde uygulanabilir üretilebilir deneyi.

<table border="0" cellpadding="0" cellspacing="0" width="1074">
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			<td height="21" style="height:21px;width:381px;">Collagenase D</td>
			<td style="width:257px;">ROCHE</td>
			<td style="width:177px;">11088882001</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>ROCHE</td>
			<td>10103586001</td>
			<td></td>
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			<td height="21" style="height:21px;">Collagenase B</td>
			<td>ROCHE</td>
			<td>11088815001</td>
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			<td height="21" style="height:21px;">Dnase</td>
			<td>ROCHE</td>
			<td>10104159001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">1X Red blood cell lysis buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
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			<td height="21" style="height:21px;">RPMI Medium 1614 1X</td>
			<td>Gibco</td>
			<td>11835-030</td>
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			<td height="21" style="height:21px;">DPBS without calcium and magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
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			<td height="21" style="height:21px;">Percoll</td>
			<td>GE Healthcare</td>
			<td>17-5445-02</td>
			<td>For density gradient centrifugation</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS &#8482; C tube&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMACS dissociator device</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use the program SPLEEN_04</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell activation cocktail (with Brefeldin A)</td>
			<td>Biolegend</td>
			<td>423303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD16/32</td>
			<td>eBioscience</td>
			<td>14-0161-81</td>
			<td>dilute 1:100</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD fixable violet dead cell stain kit</td>
			<td>Life Technologies</td>
			<td>L34955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transcription factor buffer set</td>
			<td>BD Pharmingen</td>
			<td>562725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OneComp eBeads&#160;</td>
			<td>eBioscience</td>
			<td>01-1111-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">123 count eBeads</td>
			<td>eBioscience</td>
			<td>01-1234-42</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD45 AmCyan (clone 30-F11)</td>
			<td>BioLegend</td>
			<td>103138</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CD3 PerCP-Cy5.5 (clone 17A2)</td>
			<td>BioLegend</td>
			<td>100218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17A FITC (clone TC11-18H10.1)</td>
			<td>BioLegend</td>
			<td>506910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-17F APC (clone 9D3.1C8)</td>
			<td>BioLegend</td>
			<td>517004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4 APC-Cy7 (clone GK1.5)</td>
			<td>BD Biosciences</td>
			<td>560181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8 APC (clone 53-67)</td>
			<td>eBioscience</td>
			<td>17-0081-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-bet PE-Cy7 (clone 4B10)</td>
			<td>BioLegend</td>
			<td>644823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN&#947; FITC (clone XMG1.2)</td>
			<td>BD Biosciences</td>
			<td>557724</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracellular staining and flow cytometry to identify lymphocyte subsets within murine aorta, kidney and lymph nodes in a model of hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

Protokol izin veren anjiyotensin II kaynaklı hipertansiyonu modelinde kemirgen böbrek, aort ve aort lenf düğümlerinden izole edilmiş T hücreleri yer yüzeyi ve hücre içi işaretlerinin belirlenmesi tarif edildiği. Temsilcisi sonuçları aşağıda sunulmuştur. Şekil 1, hipertansiyon uyarılması için anjiotensin II ile aşılanmış WT fare aort hazırlanan tek bir hücre süspansiyonu olarak T h?...

Watch this Scientific Journal Video about Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension at JoVE.com

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Bu makalede, tanımlamak ve hücre içi boyama ile fare böbrek, aort ve lenf düğümleri içinde mevcut fonksiyonel T lenfosit alt grupları ölçmek ve akım sitometri ayrıntılı bir metodoloji sağlar. anjiotensin II hipertansiyon modeli açıklamak adım-adım, usul ve flow sitometri temel ilkelerine ve hücre içi boyama seçildi.

Hücre içi Boyama ve Sitometrisi Hipertansiyon Modelinde Fare Aort, Böbrek ve Lenf düğümleri içinde lenfosit alt tanımak

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

intracellular-staining-flow-cytometry-to-identify-lymphocyte-subsets

Research

JoVE Journal

Immunology and Infection

18.1K Görüntüleme.  Vanderbilt University Medical Center.  Bu makalede, tanımlamak ve hücre içi boyama ile fare böbrek, aort ve lenf düğümleri içinde mevcut fonksiyonel T lenfosit alt grupları ölçmek ve akım sitometri ayrıntılı bir metodoloji sağlar. anjiotensin II hipertansiyon modeli açıklamak adım-adım, usul ve flow sitometri temel ilkelerine ve hücre içi boyama seçildi. 

Video: Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Akış Sitometri Akciğerler ve Akciğer Boşaltma lenf düğümleri Analizi fareler cerrahi olmayan intratrakeal damlatma

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

İmmünoloji ve Enfeksiyon

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Insan In Vitro Bağışıklık Dengesizlik Erken Devlet Tanıma Tarama Aracı Olarak Bastırılması

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Görüntü tabanlı Sitometrisi Tekniği Granülosit Fonksiyonu Değişiklikleri değerlendirin<em&gt; İn Vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Akış Sitometrisi ile Sıçan Böbrek gelen Makrofagların Fenotipik Karakterizasyonu

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

ile Enfeksiyon sırasında boşaltılması Lenf düğümleri içine Fare Cilt Dendritik Hücre Göç Eğitim Bir KAKE tabanlı Testi<em&gt; Mycobacterium bovis</em&gt; Basil Calmette-Guerin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Algılama<em&gt; Trypanosoma brucei</emManyetik Aktif Hücre Sorting ve Akış Sitometrisi ile&gt; Varyant Yüzey Glikoprotein Değiştirme

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Viral Örneklerin Negatif Boyanması İçin Kapsüllerin Biyokontainment İçerisinde Kullanımı

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...