私たちは、原稿の重要な読書や編集のためにH.フレミングに感謝します。この作品はMisrockポストドクトラルフェローシップ（TD）とNDSEG大学院生フェローシップ（AP）によってサポートされていました。 SNBはHHMIの研究者でもある。

1。細胞調製<ol><li>標準的な細胞培養技術を用いて継代細胞系。この実験では、OVCAR-8細胞（NCI DCTD腫瘍リポジトリ、フレデリック、MD）を用いた。 100％ - 80のコンフルエントターゲットに細胞を成長させる。 </li><li> 5分間5ミリリットルトリプシンで細胞をインキュベートし、その後、5ミリリットルRPMI培地+ FBS、トリプシンを不活性化するために追加します。 </li><li>収穫と血球のセルを数える。 5×10 7細胞/ ml、またはフラスコ当たり約200μlの目標濃度でフェノールレッド不含DMEMに再懸濁する。 </li><li>減少増殖因子マトリゲル（BD Biosciences社）15％を追加して、移植まで氷上で細胞懸濁液を保つ。 </li></ol> 2。腫瘍移植<ol><li>イソフルラン3％を使用した4週齢の雌NCR / nuマウスを麻酔し、深い麻酔を確保するために、約5分待ちます。麻酔下ながら、乾燥を防ぐために、目に獣医軟膏を使用します。 </li><li> 1ミリリットルの注射器で細胞を（腫瘍あたり100μl）をロードし、27 1を添付/ 2ゲージの針。 </li><li> 2国間後肢フランク注射部位のそれぞれについて、テントを作るとベースで細胞を注入するためにピンセットで優しく肌を持ち上げます。血を生産、あまりに深く浸透しないように注意してください。優しくはねずにシリンジをピンセットで取り除いてください。 </li><li> 2の腫瘍直径まで20日 - - 4ミリメートルに達した10毎日腫瘍の成長を監視します。 </li></ol> 3。細菌の準備<ol><li> -80℃の冷凍庫ストック又は冷蔵プレートから3ミリリットルLB培地+アンピシリン中の細菌の一晩培養を開始します。この実験では、使用されるS.ネズミチフス菌株ELH430（SL1344 PhoPQ-のaroA-）。 </li><li>午前中は、フィルタリングLBに文化1:100に希釈し、OD600 0.4に成長 - 0.6。 </li><li>スピンダウンとリンで3回洗浄する生理食塩水（PBS）は、バッファリングされ、0.1の最終OD600（1×10 7細胞/ ml程度）で再懸濁します。 </li></ol> 4。細菌インジェクション<ol><li>動物を麻酔あなたの利き手に向かって指している尾を持つその側にDポジション。麻酔下ながら、乾燥を防ぐために、目に獣医軟膏を使用します。 </li><li>温水または熱ランプを用いて尾静脈を拡張する。 </li><li> 1ミリリットルのシリンジ内細菌（マウス当たり100μlの）をロードし、90度弱の先端を曲げる。 </li><li>尾静脈とシリンジ先端を合わせ、浅い深さ（全く抵抗を感じないはず）に浸透し、細菌を注入する。成功した注射用血流の変位を観察する。 </li></ol> 5。マウスイメージング<ol><li>誘導室に動物を麻酔はその後イメージングプラットフォーム上の各マウスを置きます。麻酔下ながら、乾燥を防ぐために、目に獣医軟膏を使用します。時点との間の定量的な結果を保証するために正確な位置決めを維持する。 </li><li> IVISスペクトラムイメージングシステム（キャリパーライフサイエンス）を使用して、画像の動物。それらの間のイメージング以上1動物、場所光バリア、クロスilluを防止する場合mination。動物は、細菌の局在を確認するために、腹側の第1の結像されるべきである。 </li><li>画像の動物は背IVISの実験期間（36時間）ごとに2時間を使って。与えられたROIのタイムコースを生成します。 </li></ol> 6。細菌の生体内分布を定量<ol><li>吸収おむつで背中にCO 2と場所を使って動物を安楽死させる。 </li><li> 2後肢脇腹皮下腫瘍を露出するために動物を回します。腫瘍を分離除去するために周囲の皮膚組織をカット。ピンセットで腫瘍を保持し、逆はさみの動きを使って皮膚を除去。皮膚の大部分が分離されたときに、完全にピンセットを用いて腫瘍を取り除く。 </li><li>予め秤量マイクロチューブ内の臓器を置きます。これらのチューブの質量が大きく異なりますので、それは定量的な結果のためにそれらを事前に比較検討することが重要です。 </li></ol> 7。細菌の生体内分布を定量<ol><li>各時点で、消費税のために、腫瘍重量を量る。 </li><li> 500μlのPBS + 15％のグリセロールを追加し、組織特異Tearor（BIOSPEC）を使用してホモジナイズする。サンプル間のエタノールでホモジナイザー3回すすいでください。 </li><li>段階希釈し、細菌コロニーカウント用のプレートを生成します。単板を使用し、事前区分プレート上にプレートを複数の希釈に。 </li></ol>

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利害の衝突は宣言されていない。

この手順を用いて、腫瘍を定着細菌の増殖および遺伝子発現動態のタイムコースを生成することができる。これらの測定は、定期的にバッチ培養又はマイクロ流体デバイスにおいてインビトロで行われるが、それらはインビボで行うことがはるかに困難である。 これらの方法を適用することができるいくつかの変更がある。我々はマウスモデルを生成する?...

合成生物学は、過去10年間で急速に進んでおり、現在はエネルギーと健康に重要な問題に影響を与えるように配置されている。しかしながら、臨床分野への展開は安全上の懸念及びin vivoでの遺伝子回路の設計基準を開発するの有無によって遅くされている。加速高い衝撃の医療アプリケーションでは医療インフラ、管理されたラボ環境の外に機能する遺伝子回路、安全かつ臨床的に受け入れられる微生物ホストと直接インタフェースする方法を利用して必要になります。 菌株の数は、腫瘍において優先的に増殖する能力に起因する癌治療のために研究されてきた。これらは、C.が含まれているノビイ、大腸菌、V. cholorae、B.ロンガム 、およびS.ネズミチフス3-8。S.彼らは9-12ヒト臨床試験の数は、安全性と寛容性を示してきたようにネズミチフス菌は、特定の関心を生成しました。これらbacteri<html>最初にホストの免疫系の刺激を介して、癌細胞代謝に必要な栄養素の欠乏による抗腫瘍効果を作成することが示された。治療上の貨物の生産は後で遺伝子改変を通して追加されました。これらの研究は、腫瘍治療のための細菌の使用における重要な進歩を表しているが、既存の努力の大半は、典型的に高い投与量、オフターゲット効果、およびホスト抵抗13-16の開発の送達をもたらす高レベルの発現に依存している。 さて、合成生物学は、高度なセンシングと配信17-20を実行できる計算に設計遺伝子回路を利用することによって、プログラム可能な貨物の生産を追加することがあります。これらの回路は、腫瘍特異的な刺激を感知し、必要に応じて貨物の生産を自己調節する送達システムとして機能するように設計することができる。しかし、 生体内でこれらの回路の機能を研究することはこれまで挑戦してきました。 ve_content &quot;&gt;プラスミド、合成回路の共通のフレームワークであるため、我々は、マウスモデルを用いたin vivoでプラスミドベースの遺伝子発現のダイナミクスを特徴づけるための方法を説明しますこれらのメソッドは、タイムラプス発光イメージングと生体内分布の定量的測定を利用しています一緒に、これらのアプローチは、臨床応用のための生体内でのプラスミドベースのネットワークを研究するためのフレームワークを提供します。

<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

これらの実験の目的は、弱毒Sの増殖および遺伝子発現の動態に関する定量経時データを生成することである腫瘍の内部で成長菌細菌コロニー。 我々は、OVCAR-8（NCI DCTD腫瘍リポジトリ、フレデリック、MD）、ヒト卵巣癌細胞系の皮下注射によってマウスにおける異種移植モデルの腫瘍を発生した。 私たちは、S.の弱毒株を変換可視化2用プラスミド構成的に発現ルシフェラーゼ（luxCDABE）と： ネズミチフス菌 （SL1344 phoPQ-1 ELH430）。マウス1〜本質的に非病原性を維持しながら、これらの株は、特に腫瘍を植民地化する。 測定可能な腫瘍が確立した後、細菌は、投与量を変化させた尾静脈を介して静脈内注射した。腫瘍局在化、細菌の遺伝子発現を用いた60時間かけてリアルタイムでモニターしたvivoイメージングシステム（IVIS） であった 。各時点で、腫瘍を均質化し、切除し、そして遺伝子発現データとの相関のための細菌コロニーを定量するために播種した。 併せて、このデータは、腫瘍の内部に成長する細菌のin vivo増殖および遺伝子発現ダイナミクスの定量的尺度を与える。

このプロトコルを使用して、我々は腫瘍標的細菌のin vivo増殖および遺伝子発現ダイナミクスに関するデータを生成することができる。全体的なワークフローを図1に要約する。 第一段階では、細菌（緑）画像のレポーターとしてルミネッセンス（青）で遺伝子発現を測定するためにIVISを用いて動物を注入する。 次に、第二?...

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

これらの実験の目的は、弱毒化の増殖および遺伝子発現の動態に関する定量経時データを生成することである<em&gt; S.ネズミチフス菌</em腫瘍内部成長&gt;細菌コロニー。このビデオでは、腫瘍細胞の準備と注入、細菌の準備と注入、全動物の発光イメージング、腫瘍切除、および細菌のコロニーカウントをカバーしています。

腫瘍標的の測定成長と遺伝子発現ダイナミクス<em&gt; S.ネズミチフス菌</em&gt;バクテリア

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

measuring-growth-gene-expression-dynamics-tumor-targeted-s

Research

JoVE Journal

Immunology and Infection

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

12.1K ビュー.  Massachusetts Institute of Technology. これらの実験の目的は、弱毒化の増殖および遺伝子発現の動態に関する定量経時データを生成することである S.ネズミチフス菌細菌コロニー。このビデオでは、腫瘍細胞の準備と注入、細菌の準備と注入、全動物の発光イメージング、腫瘍切除、および細菌のコロニーカウントをカバーしています。

ビデオ: 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.

視覚化し、定量化するために蛍光タンパク質を使用しました<em&gt;クラミジア</em生細胞内&gt;液胞の成長ダイナミクス

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

免疫・感染学

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.

遺伝子発現のすべての段階でウイルス機能を定量化するためのワクレポーターウイルス

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.

感染するためのプロトコル<em&gt;線虫（Caenorhabditis elegans）</em&gt;と<em&gt;ネズミチフス菌</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.

表現型のハイスループットアッセイ<em&gt;サルモネラ菌</em&gt;ネズミチフス菌協会、侵略、およびマクロファージにおける複製

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.

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

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.

デジタルバーコードプラットフォームを使用して感染する微生物の遺伝子発現プロファイリング

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.

インフルエンザ血球凝集素抗体による Fc を介したエフェクター機能を評価する方法

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.

サルモネラの分離-大食細胞のファゴゾームを含む

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.

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

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.

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.

サルモネラ感染組織培養細胞におけるNF-κB依存性ルシフェラーゼ活性化と遺伝子発現の定量

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