Hye-Sook Oh, Joanne Morello ve Alan Collmer, Bitki Patolojisi ve Bitki-Mikrop Biyoloji Bölümü, Cornell Üniversitesi, değerli tartışmalar için teşekkür ederiz. Biz Jesse Munkvold yazı hakkında yorum için teşekkür ederim. Finansman Ulusal Bilim Vakfı Bitki Genomu Programı tarafından sağlanan, ödül sayısı DBI-0605059 (GBM).

Bölüm 1: Bitki büyüme ve bakım Tayininde kullanılan Nicotiana benthamiana tesislerinde yaklaşık 7 haftalık olmalıdır. Onlar tüm aksiller dallar ve çiçekler kaldırmak için tahlil için en az 4-5 gün önce kesilmiş olmalıdır. Ortaya çıkar sonra bitkilerin daha kolay yönetilebilir hale getirmek için yakında aksiller dalları çıkarmak için iyi bir fikirdir. Bölüm 2: Bakteriyel kültür 1. GÜN: <ol><li> Plakalar ya da kültürler tayininde kullanılan tüm suşlar için aynı anda başlatılır. Pseudomonas spp. 2 indükleyicileri KBM plakalar üzerine dondurulmuş gliserol stokları (Tablo 2) uygun antibiyotik içeren ve 3 zorluk suşları, çizgi bakteri içerir. Küçük bir miktarda bakteri plakasının ortasına yayılmış olmalıdır. Plaka yaklaşık 18-24 saat boyunca 30 ° C&#39;de inkübe edin. 30 Agrobacterium için yeni bir plaka, 2 ml LB sıvı kültürü başlatmak ve 250 rpm&#39;de gecede büyümek ° C </li></ol> 2. GÜN: <ol start="2"><li> Pseudomonas için, bakteriler sıvı KBM 150-200 ul ekleyin ve steril bir cam dağıtıcısı kullanılarak tüm plaka üzerinde bunları yaymak. Geri inkübatör plaka koyun ve bir 20-24 saat büyümesine izin. Iyi bir büyüme gösteren, bakteriyel bir plaka ertesi gün çim olmalıdır. Agrobacterium için, 20 mcM acetosyringone ile yaklaşık 5-10 ml LB ikincil kültür başlatmak ve 20 saat gece için 250 rpm, 30 büyümesine izin ° C. </li></ol> Bölüm 3: Test için hazırlık bitkiler 2. GÜN: <ol><li> Tahlil bir gün önce bitkiler 22-24 bir odaya taşınmış ° C olmalıdır, ~% 35-40 RH ve sabit ışık. </li><li> Kalın siyah bir kalem kullanarak en az 1.5 cm çapında yaprakları çevreler işaretleyin. Yaprağı başına iki ila dört çevrelerin işaretlenebilir. Çevrelerinde de aralıklı olarak ve tercihen büyük bir ven çapraz olmamalıdır. Iyi genişletilmiş yaprakları seçin. Büyük yaprakları veya dokunmatik sert veya kalın ve yaprağın alt kısımları çevrelerin işaretleme önlemek yaprakları kaçının. </li></ol> İşaretleme çevrelerin bir gün önce deneme için protokol için gerekli değildir. Ancak, test olan bitkilerin çok sayıda varsa kaydeder ve daha kolay PTI indüksiyonu ve meydan aşılar arasındaki hassas zamanlama elde etmek için yapar. Bölüm 4: PTI indüksiyonu 3. GÜN: <ol><li> P. fluorescens ve P. 10 mM MgCl 2 plaka hücreleri Hasat putida. Agrobacterium için, kültür hücreleri toplamak için santrifüj ve 10mM MgCl 2 + 10 mM MES pH 5.6 tekrar süspansiyon haline getirin. MgCl 2-MES çözümü santrifüj ve tekrar süspansiyon tekrarlayın. 600 nM hücreleri optik yoğunluk (OD) ölçün. ODS nihai gerekli değerleri Tablo 2&#39;de açıklandığı gibi. Pseudomonas sonunda 10 mM MgCl 2 ve Agrobacterium yeniden süspanse edilmelidir MgCl 2-MES çözümü ayarlayın. 25 ml toplam hacmi yaklaşık 40-50 noktalar aşılamak için yeterli. </li><li> PTI indükleyicileri 1 ml iğnesiz şırınga kullanılarak yaprakları üzerinde önceden işaretlenmiş çevrelerin içine sızmak. Bitkiler infiltre edildiği düzeni ve infiltrasyon başladı tam zamanı yazınız. </li></ol> Bölüm 5: Mücadelesi aşılama <ol><li> P. hazırlayın syringae pv. domates DC3000, 4.1 ve Tablo 2&#39;de açıklandığı gibi meydan Pst DC3000 Δ hopQ1-1 ve Ps pv. tabaci. </li><li> Indüksiyon yapıldı indükleyicisi infiltrasyon Yedi saat sonra meydan infiltrasyon, bitkilerin aynı sırayla gerçekleştirin. Genellikle, ilk aşılama dairenin çevre üzerinde bir nokta, ikinci aşılama dairenin merkezi olarak kullanılabilir. Bir yaprak üzerinde birden fazla lekeler varsa o sızıntıların çakışmadığından emin olun. </li><li> Tam CFU sayısını belirlemek için uygun antibiyotik içeren KBM veya LB plakalar üzerinde tayininde kullanılan tüm kültürlerin Plaka seri dilüsyonları. </li></ol> Bölüm 6: PTI parçalanması için Puanlama 5. Gün: <ol><li> Pst DC3000 ile itiraz edilen noktalar hücre ölümü nedeniyle ETİ görünüm için bakın. Infiltrasyon örtüşen alanı içinde hücre ölümü PTI bir dökümünü gösterir ve olumlu bir fenotip olarak atılır. </li></ol> 7. Gün: <ol start="2"><li> Pst DC3000 Δ hopQ1-1 veya Ps pv ile itiraz edilen noktalar hücre ölümü nedeniyle hastalığa görünüm için bakın. Tabaci </em&gt;. Puan bölüm 6.1 &#39;de açıklandığı gibi, aynı şekilde olumlu bir fenotip. </li></ol> Kontrol bitkiler (PTI için tehlikeye olmamalı) infiltrasyon örtüşen alanda hücre ölümüne göstermeye başladığınızda, başka gözlemler yapmayı durdurmak. Bölüm 7: Temsilci sonuçları Şekil 1 P. bir test sonucunu gösterir fluorescens meydan okuma olarak indükleyicisi ve Pst DC3000 olarak kullanılmıştır. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Şekil 1" border="0" /> Şekil 1 P. fluorescens N. üzerine sızmış benthamiana yaprakları (siyah daire) PTI ikna etmek için ve 7 saat sonra, spot Pst DC3000 (beyaz daire) ile meydan. FLS2 için susturuldu Tesisleri P. bölgede PTI bir dökümünü gösterdi fluorescens hücre ölümü görüldüğü gibi, (A) infiltre edildi. Kontrol bitkiler susturulması değil, PTI (B) indüksiyon nedeniyle örtüşen alanda hiçbir hücre ölümü gösterdi. Kırmızı oklar eksikliği veya infiltrasyon örtüşen alanda hücre ölümüne varlığı göstermektedir. Fotoğraflar, infiltrasyonların 2 gün sonra alındı. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">buraya tıklayınız</a> . <table border="1"><tbody><tr><td> PTI indükleyicisi </td><td> Hücre ölüm eliciting meydan okuma </td><td> Doğa hücre ölümü </td><td> Kod </td></tr><tr><td> Pseudomonas fluorescens 55 </td><td> Pseudomonas syringae pv domates (Pst.) DC3000 6 </td><td> ETİ </td><td> / Pf DC </td></tr><tr><td> P. putida KT2440 </td><td> Pst DC3000 </td><td> ETİ </td><td> Pp / DC </td></tr><tr><td> P. fluorescens 55 </td><td> Pst DC3000 Δ hopQ1-1 6 </td><td> Hastalık </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae pv. tabaci 11528R </td><td> Hastalık </td><td> Agro / Ptab </td></tr></tbody></table> Tablo 1: PTI uyaran ve hücre kombinasyonları ölüm eliciting PTI için hücre ölümü tayininde kullanılır mikropların. <table border="1"><tbody><tr><td> Bakteri suşu </td><td> Seçim orta </td><td> Final OD deneyde kullanılan </td><td> Sorumlu CFU / ml </td></tr><tr><td> P. fluorescens 55 </td><td> KBM ampisilin (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM ampisilin (100 ug / ml) </td><td> 0,5 </td><td> 1 x 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampisin (100 ug / ml) </td><td> 0,5 </td><td> 5 x 10 8 </td></tr><tr><td> Pst DC3000 </td><td> KBM Rifampisin (100 ug / ml) </td><td> 0,02 </td><td> 2 x 10 7 </td></tr><tr><td> Pst DC3000 Δ hopQ1-1 </td><td> KBM Rifampisin (100 ug / ml) </td><td> 0.1 100 kat seyreltme </td><td> 1 x 10 6 </td></tr><tr><td> P. syringae pv. tabaci 11528R </td><td> KBM Rifampisin (100 ug / ml) </td><td> 0.1 100 kat seyreltme </td><td> 1 x 10 6 </td></tr></tbody></table> Tablo 2: Kültür ve inokulum seviyeleri tayininde kullanılır. OD ve ilgili CFU seviyeleri spektrofotometreler farklı marka arasında değişebilir.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

Hücre ölümü tabanlı test PTI bir gen katılımı belirlemek için kullanılır. Örneğin, bir gen ilgi kaybı fonksiyon-mutantlar Bu testte kullanılarak test edilebilir. Indükleyicisi ve Tablo 1&#39;de verilen mücadele aşılar 4 kombinasyonları Out, sadece tek bir kombinasyonu bitki arka planda bir fenotip neden olabilir mümkündür. Biz FLS2 susturulması için bitkiler Pf / DC ve indükleyicisi ve meydan aşılar (Tablo 1) Pf/Q1-1 kombinasyonları PTI bir dökümünü gösterir olduğunu gözlemledik. Bu testi için çevre koşulları, Bölüm 3.1 &#39;de açıklanan olanlar mümkün olduğu kadar yakın olduğunu esastır. Düşük nem puan tahlil zorlaştırır, çok hızlı devam etmek için hücre ölümüne neden olur. Protokolde belirtilen şartlar laboratuarında işe yaramazsa, daha sonra indükleyicisi veya meydan aşılama düzeyini değiştirerek deneyin. Değiştirilmiş olabilir başka bir parametre daha kısa zaman aralıkları tahlil kontrolü bitkiler çok hızlı bir şekilde yıkmak için neden olduğu bulundu olmasına rağmen, gidiş ve meydan arasındaki zaman aralığı. Biz en az 4-5 bitkiler bir deneyde test edilmesi ve olumlu bir fenotip en az 3-4 bağımsız deneyler tekrar edilmesi önerilir.

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<td>Life Science UV/Vis Spectrophotometer</td>
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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

Çevreleri potansiyel patojen algıladıkları için, bitkiler, örüntü tanıma reseptörleri (PRRs) plazma membranlar mevcut kullanın. PRRs patojen ilişkili moleküler desenleri (PAMPs) olarak adlandırılan korunmuş mikrobiyal özelliklerini tanımak ve bu algılama, etkili olmayan patojenlerin 1,2 bitki dokularında kolonizasyonu engeller PAMP tetiklenen bağışıklık (PTI), yol açar. PTI içinde en çok çalışılan sistem FLS2-bağımlı yolak 3. FLS2 bakteriyel flagellin bir bileşeni olan PAMP flg22 tanır. Başarılı patojenlerin virülans faktörleri veya PTI bastırmak ve patojen hastalık 1 neden izin etkileyiciler sahiptir. Sırayla Bazı bitkiler effektör veya efektör tetiklenen bağışıklık (ETİ) 2 yol açar faaliyet tespit direnç genlerine sahip. Oh ve Collmer 4 değiştirilmiş PTI için bir hücre ölümü tabanlı test açıklar. Testin N. standardize benthamiana giderek bitki-patojen etkileşimi 5 çalışma için bir model sistemi olarak kullanılmaktadır. PTI, bir yaprak patojen olmayan bakteri suşunun infiltrasyonu indüklenir. Yedi saat sonra, bir bakteri suşunun da hastalığa neden ya da ETİ orijinal infiltrasyon bölge örtüşen bir alana sızmış harekete geçirir. Ilk infiltrasyonu ile indüklenen PTI görünümünü ikinci bir meydan okuma infiltrasyon nedeniyle hücre ölümü geciktirmek veya önlemek mümkün. Tersine, inokülasyon örtüşen alanda hücre ölümüne görünümünü PTI bir dökümünü gösterir. PTI ve meydan aşılar indükleyicileri dört farklı kombinasyon standardize edilmiştir (Tablo 1). Tahlil sessiz N. üzerinde test edildi benthamiana bitkiler kontrolü ve PTI geliştirmek için yeteneklerini tehlikeye gireceği tahmin edilmiştir FLS2 için susturuldu bitkiler olarak görev yaptı.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

PTI Nicotiana benthamiana bitkiler için bir hücre ölümü tabanlı test açıklanmıştır.

Patojen İlişkili Moleküler Desen (PAMP) Triggered Tesisleri Bağışıklık (PTI) için Testi

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

assay-for-pathogen-associated-molecular-pattern-pamp-triggered

Research

JoVE Journal

Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K Görüntüleme.  Boyce Thompson Institute for Plant Research. PTI Nicotiana benthamiana bitkiler için bir hücre ölümü tabanlı test açıklanmıştır.

Video: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Seçimi Plasmodium falciparum İnsan Beyninin endotel hücreleri Cytoadhesion için Parazitler

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

İmmünoloji ve Enfeksiyon

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Kayma Stres İnsan Karaciğer Sinüzoidal Endoteline altında Koşullarına Lökositer İşe Alım Eğitim için Bir Akış Adezyon Deneyi

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Yüksek Kapasiteli saflaştırılması<em&gt; Plasmodium falciparum</emOpsonizing Antikor Analizlerde kullanım için&gt; merozoitler

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Floresans tabanlı Nöraminidaz İnhibisyonu Tahlili antivirallerin nöraminidaz inhibitörü Class Nezle Virüslerinden eğilimi değerlendirilebilir

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Fare modelleri Helicobacter enfeksiyonu ve gastrik patolojiler

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Viral enfeksiyon ve analiz Drosophila Melanogaster ana bilgisayar virüs etkileşimin kurulması

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Çift iplikçikli RNA ve desen tanıma reseptörleri negatif anlamda RNA virüsü enfeksiyonu confocal görüntüleme

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;