この原稿のためのビデオ編集と彼女の支援のためのトランスレーショナル科学UTMB-NIHの助成金1UL1RR029876-01とアリーシャプレイザー研究所。

1。動物の準備<ol><li>動物の到着：動物バイオセーフティレベル2（ABSL2）施設への到着時に、動物に、新しい環境に順応するために2日以上を可能にします。この休息期間に続いて、自分の健康と全体的な物理的な外観を評価するために動物を検査する。 <ol><li>蛍光レポーターを利用するとき、それはGIの自家蛍光とバック​​グラウンド信号の量を制限するためにアルファルファフリーダイエット上のすべての動物の科目を配置することが重要です。 </li></ol></li><li>動物を剃る：撮像中に頭蓋領域から検出された生物発光信号を改善するために、すべての動物の頭を剃る。酸素ガスの混合物で動物を麻酔し、イソフルラン気化。動物は完全に麻酔したら、頭を剃ること電気バリカンを使用しています。一度シェービングを終え、ケージに動物を戻すと麻酔の影響から完全に回復のために守ってください。 </li><li>バイオメディックデータシステム（BMDS）トランスポンダ挿入（動物は完全に麻酔をかけている間、マウスのシェービング後に場所を取るために）：低侵襲の手段のため事前にプログラムされBMDS無線トランスポンダは、各マウスの背部皮下に温度インプラントを追跡する。これらのトランスポンダは、無線識別と温度記録が可能になります。トランスポンダの移植後、傷口に獣医グレードの組織接着剤を適用します。 </li><li>ベースラインコレクション：感染する前に、すべての動物のためのベースライン温度と重量を記録する。マウスは麻酔下にある間眼窩（RO）のブリードスループラーク減少中和試験（PRNT）分析のために血液を採取します。採血後、潜在的な二次的細菌感染を減らすために獣医抗生物質眼軟膏を適用します。このベースラインコレクションは、動物がマウスに起因する応力に麻酔下に配置​​された回数を考慮して完了する必要があります。今後のROのコレクションマウスを麻酔下にまだある間、hould、イメージング後に完了する。 ROから収集された最大血は200μl程度になるはずです。 </li><li>感染症：前述のように麻酔下でマウスを配置します。鼻腔感染によって緩衝生理食塩水（PBS）で希釈されたリン酸を40μlの総体積で5×10 6〜5×10 7 pfuの TC83-ルシフェラーゼの用量を用いた鼻腔内経路を介して接種する。感染後、彼らの住宅ケージ内にマウスを置くと回復を観察します。 </li></ol> 2。動物イメージング 免責事項：すべての回で彼らの住宅の単位、生物学的安全キャビネット（BSC）、またはXIC封じ込めボックス内でマウスをおいてください。このプロトコルのための承認されたのBSCは、クラスIIタイプB1またはB2です。 <ol><li> 15 mg / mlの濃度で腹腔内（IP）ルシフェリンの体重1グラム当たり10μlのすべての動物を注入します。 <ol><li> Ampligenを受けたマウスはinjecteされるそれらのグループに基づいて-4時間後に感染または48時間後に感染症で12.5 mg / kg体重の用量でDのIP。 </li></ol></li><li>ルシフェリンの注射の前に、XIC-3封じ込めボックス内の適切なフィットを保証するために、3つのグループにマウスを分ける。 <ol><li>すべての画像は、手順全体で角膜の乾燥を防ぐために、眼軟膏剤/潤滑剤を使用しています。 </li></ol></li><li> LivingImageソフトウェアを開き、イメージングボックスを準備するために初期化を押し、設定した自動保存、自動（自動で露光時間がLivingImageソフトウェア3.2およびそれ以降の新機能である）への露光時間、Cへのフィールドを表示したり、開くことがフィルター;フィールドビューのあなたがイメージングであるマウスの数に基づいて行われ、その他のイメージングプロジェクトのために必要に応じてフィルタを最適化することができる。 </li><li>エアチャコールフィルタの期限が切れていないことを確認し、酸素タンクがいっぱいで、イソフルランリザーバが満たされている。私はXIC-3に電気テープの小さなストリップを置き撮像時に動物の動きを減らすことを支援ゾル化ボックス。 </li><li> 3〜5分間（ふたが開いている）イソフルラン誘導チャンバ内にセクション2.1と場所で説明したようにルシフェリンのIPをマウスに注入します。 </li><li> 5分後、誘導チャンバーの蓋を閉めるとイソフルランのフローを開始する。麻酔は、約2.0リットル/分に設定酸素流量で約3〜5％で運転される。一度完全に無意識のさらに30秒待つとXIC-3分離ボックスにマウスを移す。この手順としてイソフルランの掃気安全が誘導室（IVISユニットは独自のパッシブフェアキャニスターが含まれているとXIC-3封じ込めボックスが直接イン/アウトのソケットに接続してから、イソフルランの損失も含まれるために利用されてバイオセーフティキャビネットができますを確認します）が含まれている麻酔の流れを確保しています。 <ol><li>添付イソフルランコネクタ付きXIC-3分離ボックスにチップ/グループ数に基づいてマウスを転送dが開きます。彼らは静かにテープストリップで休んで頭と胸骨の横臥にレイアウトされているので、マウスを置きます。 </li></ol></li><li> cavicideエタノール、スプレー手とXIC-3の底部とXIC封じ込めボックスを拭うと、IVISイメージング室に移す。一度撮像部内側封じ込めボックスに麻酔を再接続します。 </li><li>後のルシフェリンの注射後10分、リビングイメージソフトウェアを使用して画像をマウス。 </li><li>開いたフィルターを使用した初期画像に続いて、配列分析および生物発光ホタルルシフェラーゼのイメージウィザードのセットアップを使用してDLIT 3次元イメージングを開始する。このイメージングプロセスは4-5分かかりますし、目的のマウスの数に関連する視野で完了する必要があります。 <ol><li> 生体外分析は、脳の可能性は、次剖検およびコレクションです。 、滅菌シャーレに脳を置き上に株式ルシフェリンの50から100μlを垂れると、IMA内の場所ボックスを変更する。 </li></ol></li><li> DLITイメージングの完了時に15から17分後の感染で第二のオープンフィルタ画像を完成させます。配列解析が切れていることを確認して、ビューとフィルタ設定のフィールドは、以前の設定に戻ります。 </li></ol> 3。マウスおよびデータ解析を返す<ol><li>イソフルラン気化器の電源をオフにし、バイオセーフティキャビネットにバックXIC封じ込めボックスを転送します。 </li><li>彼らの住居ユニット内に戻るマウスを置くと、背面筐体ラックの適切な消毒剤と場所と外観を吹きかけ、上部を閉じます。 <ol><li>マウスは完全に麻酔から回復していることを確認します。 </li></ol></li><li>撮像が完了するまでの実験が必要であるように上記の手順を繰り返します。 </li><li>は、BSCを消毒装置をシャットダウンして、さらに分析するために外部の研究室の場所にすべてのデータを転送します。 </li><li>データを分析し、IVIS結果UTIに基づいて3次元画像を生成LivingImageソフトウェアをlizing。 <ol><li>画像が適切に指向していて、照明/カラーバランスが設定されていることを確認します。ツールパレット内のオプションは、画像調整、修正、画像情報、およびROIツールが含まれています。 </li><li>場所に基づいて、特定の信号強度を識別するために、ROIの形状を利用しています。 </li><li> 、マウス本体を強調するために表面形状を再構成3D DLIT再建を初期化し、地形復興に臓器マップを設定するには、線形近似を使用しています。 </li></ol></li><li>さらにウイルスの滴定分析は血液や臓器のコレクションを介して完了することができます。本研究では滴定を改変イーグル培地（MEM）で脳組織の均質化を経て完成しました。ホモジネートを段階希釈し、我々は1時間Vero細胞の6ウェルプレートを感染させた。細胞は1.5％agarose/2xMEMオーバーレイで覆われ、37℃で2日間インキュベートした。細胞を30分間、10％ホルマリンで固定して染色したクリスタルバイオレット。 </li></ol>

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特別な利害関係は宣言されません。

このプロトコルは 、in vivo で解析するための撮像面をカバーしていますが、それは今後の研究のための重要な要因として、生物発光ベクトルを認識することは重要である。ルシフェラーゼの発現用ベクターとしてTC83当社の利用、VEEVの弱毒ワクチン株は、酵素を多量に前述の1〜4としてCNSにおけるウイルスの高い複製速度に起因する生産されていることを保証します。野?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> D-ルシフェリン</td><td></td><td></td><td></td></tr><tr><td>イソフルレン</td><td></td><td></td><td></td></tr><tr><td> Xenogen IVISシステム（スペクトラム） </td><td>キャリパーライフサイエンス</td><td></td><td></td></tr><tr><td> XGI-8ガス麻酔システム</td><td>キャリパーライフサイエンス</td><td></td><td></td></tr><tr><td> XIC-3封じ込めボックス</td><td>キャリパーライフサイエンス</td><td></td><td></td></tr><tr><td> LivingImage 4.0ソフトウェア</td><td>キャリパーライフサイエンス</td><td></td><td></td></tr><tr><td>テレメトリ/識別チップ</td><td>バイオメディックデータシステムズ</td><td> IPTT-300 </td><td>動物IDとテンパーature </td></tr><tr><td> 26 GX 3月8日 &quot;針のBDインテグラ1ミリリットル結核シリンジ</td><td>フィッシャー·サイエンティフィック</td><td> 305279 </td><td></td></tr><tr><td>獣医ボンド組織接着剤</td><td>フィッシャー·サイエンティフィック</td><td> NC9259532 </td><td></td></tr><tr><td> Vetropolycin眼科用軟膏</td><td>ウェブスター獣医製品</td><td> 78444656 </td><td></td></tr><tr><td>ダルベッコのリン酸緩衝生理食塩水1Xバッファ付き</td><td>インビトロジェン</td><td> 14190-144 </td><td></td></tr><tr><td> BMDSチップリーダー</td><td>バイオメディックデータシステムズ</td><td> DAS-7007S </td><td></td></tr><tr><td> DAS-HOSTのソフトウェア</td><td>バイオメディックデータシステムズ</td><td></td><td>プローブ情報をダウンロードするために使用</td></tr></tbody></table>

in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus

イメージング技術の近代的な進歩は、ウイルス研究が達成されるようにさらなる開発と改良を奨励しています。当初はヒュームの3R（リデュース交換、還元、リファインメント）でラッセルとBurchによって提案された、科学研究における動物モデルの利用が科学的正確さとスピードを向上させながら、動物の使用量を削減する新たな方法論を識別するために、一定の圧力の下にある。ヒュームのプリンシパルへの主要な課題は、しかし、動物の疾病の罹患率と全体の数字を削減しつつ、研究は統計的に正確であることを確認する方法です。ワクチンの有効性の研究は、現在、統計的に有意であると見なされるためには、動物の多数を必要とし、多くの場合、免疫防御の識別のための高い罹患率と死亡率のエンドポイントにつながる。我々は徐々にリファレンスによって、中枢神経系（CNS）の侵入を追跡するためにホタル生物発光酵素と一緒にin vivoイメージングシステム（IVIS） で利用マウスモデルにおけるphaliticウイルス。典型的には、病気は、比較的ゆっくりと進行する、しかしウイルス複製は、特に中枢神経系内で、迅速であり、多くの場合、致命的な結果につながることができます。 TC83-Lucを用いたマウスの鼻腔内感染後、弱毒ベネズエラウマ脳炎ウイルス株は、ルシフェラーゼ遺伝子を発現するように改変、我々は、少なくとも3日臨床疾患の症状の開発前に脳内でのウイルスの複製を視覚化することができます。我々は臨床症状が発現する前に速やかにTC83-Lucの感染症に対する治療薬およびワクチンの保護を識別することができるキー脳炎疾患発症のエンドポイントとして中枢神経系への浸潤を利用。 IVIS技術により、我々は動物数および罹患率を低減しつつ、薬物治療やワクチンの迅速かつ正確なテストを実証することができます。

ウイルス複製が中央CNS（ 図1）に鼻の領域から移動すると、遺伝的に改変されたウイルスで、TC83-ルシフェラーゼ、我々は、生物発光信号強度の増加を見た。高いウイルス複製速度のために、我々はベクトルにベクトルや動物の免疫応答に依存する生物発光信号のハイレベル（ 図2A）を参照してください期待しています。我々は、CNS内のウイルス複製のピ...

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In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

小説としてルシフェラーゼおよびin vivoイメージングシステム（IVIS）を活用することで臨床開発が発生する前に病気のエンドポイントを識別することを意味します。 IVISは、私たちは今後の研究のために、より正確な疾患モデルを提供し、リアルタイムで複数の日にわたって脳炎ウイルスの侵入を可視化することができました。また、私たちは高速に現在利用されている動物モデルよりも抗ウイルス薬やワクチンの潜在的な保護機能を識別することができました。複数の時点にわたって個々の動物を利用する能力は、病気の研究のより人間的な、より科学的な手段を確保し活用動物に減少動物要件、コスト、および全体的な罹患率を保証します。

神経侵襲性脳炎ウイルスのin vivoイメージングシステム（IVIS）検出で

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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Immunology and Infection

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

20.5K ビュー.  University of Texas Medical Branch. 小説としてルシフェラーゼおよびin vivoイメージングシステム（IVIS）を活用することで臨床開発が発生する前に病気のエンドポイントを識別することを意味します。 IVISは、私たちは今後の研究のために、より正確な疾患モデルを提供し、リアルタイムで複数の日にわたって脳炎ウイルスの侵入を可視化することができました。また、私たちは高速に現在?...

ビデオ: In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

ライブホストのトランスジェニックリーシュマニア原虫のin vivoイメージング

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

免疫・感染学

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

水系でのウイルス産生率を予測

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

インフルエンザウイルスのためのハイスループット検出方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

豚からのインフルエンザAウイルスの検出および単離のための鼻ワイプ

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

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 ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

組換えプロトタイプ フォーミー ウイルス インテグラーゼの浄化中にヌクレアーゼの混入の検出と除去

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...