Biz yazının eleştirel okuma ve düzenleme için H. Fleming teşekkür ederim. Bu çalışma Misrock Doktora Sonrası Bursu (TD) ve NDSEG yüksek lisans bursu (AP) tarafından desteklenmiştir. SNB bir HHMI Araştırmacı olduğunu.

1. Hücre Hazırlanması <ol><li> Standart hücre kültürü teknikleri kullanılarak geçişi hücre çizgileri dahildir. Bu deneyde, OVCAR-8 hücreleri (NCI DCTD Tümör Deposu, Frederick, MD) kullanılır. 100% - 80 confluency bir hedef hücreleri büyütün. </li><li> 5 dakika boyunca 5 ml tripsin ile inkübe hücreleri, ardından + FBS tripsin inaktive 5 ml RPMI ortamı ilave edin. </li><li> Hemasitometre üzerinde hasat ve sayısı hücreleri. 5 x 10 7 hücre / ml ya da şişesi başına yaklaşık 200 ul bir hedef konsantrasyonunda kırmızı fenol içermeyen DMEM içinde süspanse edin. </li><li> 15% daha az büyüme faktörü Matrigel (BD Biosciences) ekleyin ve implantasyon kadar buz üzerinde hücre süspansiyonu tutun. </li></ol> 2. Tümör İmplantasyonu <ol><li> Izofluran% 3 ile 4 haftalık dişi NCR / Nu fareler anestezi ve derin anestezi sağlamak için yaklaşık 5 dakika bekleyin. Anestezi altında iken kuruluğu önlemek için gözleri veteriner merhem kullanın. </li><li> 1 ml şırınga içinde hücreleri (hem tümör başına 100 ul) yerleştirin ve 27 1 takmak/ 2 iğne. </li><li> İki lider, karşılıklı arka yan enjeksiyon sitelerin her biri için, bir çadır yapmak ve tabanda hücreleri enjekte forseps ile cildi nazikçe kaldırın. Kan üreten, çok derin nüfuz dikkat edin. Hafifçe sıçrama olmadan şırınga kaldırmak için cımbız kullanın. </li><li> 2 arasında bir tümör çapı kadar 20 gün - 4 - mm ulaşıldığında 10 günlük tümör büyümesini izlemek. </li></ol> 3. Bakteri Hazırlık <ol><li> Bir -80 C derin dondurucu hisse senedi veya soğutulmuş plaka 3 ml LB medya + ampisilin bakteri bir gecede kültürü başlatın. Bu deneyde, S. kullanılan typhimurium suşu ELH430 (SL1344 PhoPQ-aroA-). </li><li> Sabah, filtre LB içine kültür 1:100 sulandırmak ve OD600 0,4 büyümeye - 0.6. </li><li> Aşağı Spin ve fosfat 3 kez yıkayın tuzlu su (PBS) tamponlu ve 0.1 bir son OD600 (1 x 10 7 hücre / ml) de tekrar süspansiyon. </li></ol> 4. Bakteri enjeksiyonu <ol><li> Bir hayvan anestezisikuyruk da dominant el doğru bakacak şekilde yan d konumu. Anestezi altında iken kuruluğu önlemek için gözleri veteriner merhem kullanın. </li><li> Ilık su veya ısı lambası kullanarak kuyruk ven dilate. </li><li> 1 ml şırınga içinde bakteri (fare başına 100 ul) yükleyin ve 90 dereceden sadece daha az ucu bükün. </li><li> Kuyruk ven ile şırınga ucu aynı hizaya getirin, sığ bir derinliğe (direnç hissetmelisiniz) de nüfuz ve bakteri enjekte. Başarılı bir enjeksiyon için kan akımını değiştirme dikkat edin. </li></ol> 5. Fare Görüntüleme <ol><li> Indüksiyon odasında hayvanların anestezisi sonra görüntüleme platformu üzerinde, her bir fare yerleştirin. Anestezi altında iken kuruluğu önlemek için gözleri veteriner merhem kullanın. Zaman noktaları arasında kantitatif sonuçları elde etmek için hassas konumlandırma koruyun. </li><li> IVIS Spectrum görüntüleme sistemi (Kaliper Yaşam Bilimleri) kullanarak görüntü hayvanlar. Aralarında görüntüleme 1&#39;den fazla hayvan, yer ışık bariyerleri çapraz illu önlemek için iseğerlendirmesi. Hayvanlar bakteri lokalizasyonu onaylamak için ventral tarafta ilk görüntülü olmalıdır. </li><li> Görüntü hayvan dorsal deney (36 saat) süresince IVIS her 2 saat kullanarak. Belirli bir yatırım getirisi için bir süre ders oluşturun. </li></ol> 6. Bakteriyel biolojik dağılım miktarının <ol><li> Emici bebek bezi üzerinde arkasında CO 2 ve yer kullanarak hayvanlar euthanize. </li><li> Iki arka yan subkutan tümörler ortaya çıkarmak için hayvan çevirin. Tümörler ayırmak ve kaldırmak için, etrafını saran dokulardan kesin. Cımbız ile tümör Holding, bir ters makas hareketi kullanarak cilt kaldırmak. Cilt çoğunluğu ayrılmış zaman, tam cımbız kullanarak tümör çıkarmak. </li><li> Önceden tartılmış mikrosantrifüj tüpleri içinde organları. Bu tüplerin kitle önemli ölçüde değişebilir çünkü nicel sonuçlar için bunları önceden tartmak önemlidir. </li></ol> 7. Bakteriyel biolojik dağılım miktarının <ol><li> Her zaman noktası, tüketim ve tümörler tartın. </li><li> 500 ul PBS +% 15 gliserol ekleyin ve bir Doku-Tearor (BioSpec) kullanarak homojenize. Örnekleri arasında etanol ile homojenleştirici 3 kez yıkayın. </li><li> Seri seyreltme ve bakteri kolonisi sayımı için plaka oluşturun. Tek bir tabak kullanımı ön kesitli plakalar üzerinde plaka birden fazla seyreltme için. </li></ol>

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Çıkar çatışması ilan etti.

Bu prosedür kullanılarak, böylece tümörü kolonize bakteri büyümesi ve gen ekspresyonu dinamikleri için zaman içerisinde elde edebiliyoruz. Bu ölçümler rutin parti kültür ya da mikroflüidik cihazlar, in vitro olarak gerçekleştirilir olsa da, in vivo olarak gerçekleştirmek için çok daha zordur. Bu yöntemler uygulanabilir çeşitli değişiklikler vardır. Bizim fare modelleri oluşturmak için OVCAR-8 hücre kullanılır iken, diğer hücre hatları, bi...

Sentetik biyoloji son on yılda hızla ilerlemiştir ve şimdi enerji ve sağlık önemli sorunları etkisi konumlandırılmış. Ancak, klinik alana genişleme güvenlik endişeleri ve in vivo genetik devrelerinde için tasarım kriterleri geliştirme bir yokluğu ile yavaşladı edilmiştir. Yüksek darbe tıbbi uygulamaları hızlandırılması tıbbi altyapı, kontrollü laboratuar ortamı dışında işlev genetik devreleri, ve güvenli ve klinik-kabul mikrobiyal bilgisayarlar ile doğrudan arabirim yöntemleri kullanarak gerektirecektir. Suş tümörlerde tercihen büyümeye yeteneklerine bağlı olarak kanser tedavisi için incelenmiştir. Bu C eklemiş novyi, E. coli, V. cholorae, B. longum, ve S. typhimurium 3-8. S. onlar 9-12 insan klinik çalışmalarda bir dizi güvenlik ve hoşgörü sergilenen gibi typhimurium özel bir ilgi yarattı. Bu bakteri<html> Bir ilk konak bağışıklık sisteminin uyarılması yoluyla ve kanser hücre metabolizması için gerekli besinlerin tükenmesi ile bir anti-tümör etkileri oluşturmak için gösterilmiştir. Tedavi kargo üretimi daha sonra genetik değişiklikler ile eklendi. Bu çalışmalar tümör tedavileri için bakteri kullanımı önemli ilerlemeler temsil ederken, mevcut çabalar çoğunluğu genellikle çok yüksek dozlarda, hedef dışı etkiler ve ana 13-16 direnç gelişimi teslimat ile sonuçlanan yüksek düzeyde ekspresyon yararlanmıştır . Şimdi, sentetik biyoloji gelişmiş algılama ve dağıtım 17-20 gerçekleştirebilirsiniz hesaplama tasarlanmış genetik devreleri kullanarak programlanabilir yük üretim ekleyebilirsiniz. Bu devreler, tümör spesifik uyaran ve gerektiğinde kendi kendini düzenleyen kargo üretim anlamda dağıtım sistemleri olarak hareket için dizayn edilebilir. Ancak, in vivo olarak, bu devrelerin fonksiyonu çalışmalarında şimdiye kadar zor olmuştur. plazmid sentetik devreleri için ortak bir çerçeve olan ve_content &quot;&gt; bu yana, bir fare modeli kullanılarak in vivo plazmid tabanlı gen ifadesinin dinamiklerini karakterize etmek için bir yöntem açıklanmaktadır. Bu yöntemler zaman atlamalı lüminesans görüntüleme ve biodistribution kantitatif ölçümü kullanmaktadır. birlikte, Bu yaklaşımların klinik uygulamalar için in vivo plazmid tabanlı ağlar eğitim için bir çerçeve sağlar.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>BD Biosciences</td>
 <td>309602</td>
 <td></td>
 </tr>
 <tr>
 <td>3/10 cc Insulin Syringe</td>
 <td>BD Biosciences</td>
 <td>309301</td>
 <td></td>
 </tr>
 <tr>
 <td>PrecisionGlide Needle </td>
 <td>BD Biosciences </td>
 <td>301629</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 (1X), 
 liquid, with L-glutamine</td>
 <td>Invitrogen</td>
 <td>11875-119 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sigma Aldrich</td>
 <td>A0166-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>LB Agar Broth</td>
 <td>Sigma Aldrich</td>
 <td>L2897</td>
 <td></td>
 </tr>
 <tr>
 <td>Luria-Bertani broth</td>
 <td>BD Biosciences</td>
 <td>244610</td>
 <td></td>
 </tr>
 </tbody>
</table>

measuring growth and gene expression dynamics of tumor-targeted s. typhimurium bacteria

Bu deneylerin amacı, hafifletilmiş S. büyümesi ve gen ekspresyonu dinamikleri üzerinde niceliksel zamana bağlı verilerini oluşturmak için bir tümörleri içinde büyüyen typhimurium bakteri kolonileri. Bir insan yumurtalık kanseri hücre hattı deri altına enjeksiyon, OVCAR-8 (NCI DCTD Tümör Deposu&#39;ndan, Frederick, MD). Farelerde modeli ksenograft tümörler oluşturulan Biz, S. zayıflatılmış suş transforme görselleştirme 2 plazmid yapıcı şekilde tanımlanmış lusiferaz (luxCDABE) ile: typhimurium bakteriler (SL1344 phoPQ-1 ELH430). Fare 1, esas olarak non-virulent kalarak Bu suşlar, özellikle tümörler kolonize olur. Ölçülebilir tümörler tesis edildiğinde, bakteri dozaj değişen kuyruk damarı yoluyla venöz içinden enjekte edildi. Tümör-lokalize, bakteri gen ekspresyonundaki kullanarak 60 saatlik bir süre boyunca gerçek zamanlı olarak izlenmiştirin vivo görüntüleme sistemi (IVIS) içinde. Her zaman noktasında, tümörler homojenize eksize ve gen ekspresyon verileri ile korelasyon için bakteri kolonileri ölçmek için kaplandı. Birlikte, bu veriler tümörler içinde büyüyen bakterilerin in vivo büyüme ve gen dinamikleri içinde bir nicel ölçü verir.

Bu protokol kullanarak, tümör hedef bakteri in vivo büyüme ve gen dinamikleri veri elde edebiliyoruz. Genel akışı Şekil 1 &#39;de özetlenmiştir. İlk aşamada, biz lüminesans (mavi) bir muhabir olarak ile gen ekspresyon ölçmek için IVIS kullanarak hayvan bakteri enjekte (yeşil) ve görüntü. Daha sonra, ikinci aşamada, biz tüketim, homojenize ve koloni sayımı için plaka tümörler tümör büyüyen bakteri s...

Watch this Scientific Journal Video about Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria at JoVE.com

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

Bu deneylerin amacı, zayıflatılmış büyümesi ve gen ekspresyonu dinamikleri üzerinde niceliksel zamana bağlı verilerini oluşturmak için bir<em&gt; S. typhimurium</emTümörleri içinde büyüyen&gt; bakteri kolonileri. Bu video tümör hücre hazırlama ve implantasyonu, bakteri hazırlanması ve enjeksiyon, tam hayvan lüminesans görüntüleme, tümör eksizyonu ve bakteri kolonisi sayma kapsar.

Tümör Hedefli ölçülmesi Büyüme ve Gen İfade Dynamics<em&gt; S. Typhimurium</em&gt; Bakteri

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

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Immunology and Infection

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

12.0K Görüntüleme.  Massachusetts Institute of Technology. Bu deneylerin amacı, zayıflatılmış büyümesi ve gen ekspresyonu dinamikleri üzerinde niceliksel zamana bağlı verilerini oluşturmak için bir S. typhimurium bakteri kolonileri. Bu video tümör hücre hazırlama ve implantasyonu, bakteri hazırlanması ve enjeksiyon, tam hayvan lüminesans görüntüleme, tümör eksizyonu ve bakteri kolonisi sayma kapsar.

Video: Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Görselleştirme ve Miktarının Belirlenmesi için floresan proteinleri kullanarak<em&gt; Chlamydia</em&gt; Vakuol Büyüme Dinamikleri Yaşayan Hücrelerde

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

İmmünoloji ve Enfeksiyon

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Gen İfade Tüm Dönemlerinde Viral Fonksiyonu sayısallaştırabilmek için vaksiniya Reporter Virüsler

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Bulaştırmak için bir protokol<em&gt; Caenorhabditis elegans</em&gt; Ile<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Fenotipinin yüksek verim Deneyi<em&gt; Salmonella enterica</em&gt; Typhimurium Derneği, Invasion, ve Makrofagların Çoğaltma

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Fare Periton Makrofagların izolasyonu Toll-benzeri reseptörler Uyarım üzerine Gen İfadesi Analizi Yapacak

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

Dijital Çizgi-kodlama Platform&#39;u kullanarak bulaşmasını Mikroplar Gen İfadesi profil

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Bakterilerde Hücre Altı Protein Lokalizasyonu ve Hücre Morfolojisi Değişikliklerini Araştırmak Için Canlı Hücre Floresan Mikroskobu

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NF-&amp;#954;B-dependent Luciferase Activation and Quantification of Gene Expression in &lt;em&gt;Salmonella&lt;/em&gt; Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Salmonella Enfekte Doku Kültür Hücrelerinde NF-κB bağımlı Luciferase Aktivasyonu ve Gen Ekspresyonunun Nicelleştirilmesi

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...