著者らは、NIH 3R01AI080289 - 02S1、およびNIH / NIAID 2P30AI060354 - 07の下でエイズ研究員フェローシップのためのハーバード大学のセンターからの資金調達を認識したい。

1。文化食細胞<ol><li>文化THP - 1 Tフラスコに固定懸濁液として10％ウシ胎児血清を添加したRPMI 1640で10セル。細胞密度は、FcγRの発現とアッセイの性能の一貫したレベルを維持するために、0.5 × 10 6 / mL以下に維持されるべきである。 </li></ol> 2。ビオチン化抗原を準備<ol><li>製造元の指示に従って、目的の標的抗原をビオチニル化するために必要なスルホ- NHS LCビオチン試薬キットの量を計算します。 </li><li>水中でのビオチン化試薬を溶解し、すぐに第一級アミンを含まないバッファーで抗原に計算した金額を追加してください。反応が時折混ぜ、室温で1時間進行することができます。 </li><li>タージェを維持するために適切な分子量のカットオフのアミコン遠心濃縮装置に緩衝液交換によって過剰、非抱合型ビオチンを削除します。T抗原。濃縮ユニットの上部チャンバーにサンプルを追加し、15 mLの充填ラインへの総ボリュームを起動するPBSを加える。約1.5 MLSにボリュームをダウンさせるために4000 × gで遠心する。この処理を繰り返すと、二回ビーズに抗原の最大塗膜を確保するための遊離ビオチンの99％が削除されます。 </li></ol> 3。抗原飽和ビーズを準備<ol><li>高速遠心機でスピンダウン後、1 mlの0.1％PBS - BSAで二回1ミリメートル蛍光ニュートラビーズ100μlを洗う。再懸濁し、10のチューブに100μlのPBS - BSA、および一定量のビーズを洗浄した。 </li><li>ビーズを飽和させる抗原のコーティングの条件を決定するために、二倍のステップでビオチン化抗原の濃度を変化させて洗浄したビーズ懸濁液10 ulを組み合わせる。ローテーター上でマイクロ遠心チューブに4℃で一晩インキュベートする。 </li></ol>注：ビーズの彩度を実験的に決定する必要があります。これを達成することができるビーズが続いてコントロールモノクローナル抗体でオプソニンしているときに最大の食作用をもたらすビードコーティングの条件を識別することによって。 <ol start="3"><li>ビーズのペレットになるまで高速で1 mLのPBS - BSAと遠心で洗浄して非結合抗原を削除します。 PBS - BSAを削除して、繰り返します。 </li><li> PBS - BSAに1 mlの最終容量に再懸濁し、抗原をコーティングしたビーズ。ビーズは4℃の週に使用する° Cの前まで保存することができます。 </li></ol> 4。抗体のサンプルを準備する<ol><li>臨床抗体のサンプルは、メーカーの指示に従ってメロンゲルのIgG精製キットを用いて血漿から精製することができる。精製したIgGは4℃で使用直前まで保存することができます。 </li></ol>注：ヒトサンプルを扱うに関する適切な対策を常に取られている必要があります。 <ol start="2"><li> A280の吸光度で精製抗体の濃度を決定し、PBSで1 mg / mlにサンプルを希釈する。 </li><li>置かれているものを準備PBSで1 mg / mlまで希釈して、IVEと負のモノクローナルコントロール抗体。 </li></ol> 5。実験をめっき<ol><li>ボルテックスで上記で調製した洗浄、抗原飽和ビーズ溶液を再懸濁し、そして丸底96ウェルプレートの各ウェルに10μlを移す。ケアは、継続的に蛍光ビーズの等しい数を確保するためにビーズ懸濁液を撹拌するために注意しなければなりません各ウェルに添加されています。 </li><li>各抗体の用量 - 反応曲線を作成し、各ウェルに関心の抗体の濃度を変化させて追加します。最適な濃度が存在する抗体の力価に応じて、サンプル間で異なりますが、0.01から100μg/ mlの最終濃度の範囲は、良好なカバレッジを提供し、関心の濃度範囲の同定が可能になります。抗体を20μlのより大きくないボリュームで追加されていることを確認します。 </li><li> ° Cの抗体がビーズをオプソニン化するために可能にするために37℃で2時間ビーズと抗体のサンプルをインキュベートする。&lt;/ LI&gt; <li> 2.5 × 10 5細胞/ ml、でのTHP - 1細胞の懸濁液を調製し、各ウェルに5 × 10 4 THP - 1細胞の合計で、各ウェルにこの懸濁液200μlを加える。 </li><li> 37℃で一晩インキュベート℃、5％固定インキュベーター内でCO 2、、重力を経由してペレットにできるように、細胞とビーズ。 </li></ol>注：ノイズへの信号が最適なビーズを決定することによって改善する場合があります：抗体：THP - 1特定の抗原と抗体ソースの細胞比。 6。フローサイトメトリー解析<ol><li>各ウェルの細胞ペレットを乱さないように注意してから上清100μlを削除します。サイトカイン分泌の決定または他の分析が望まれる場合上清を保存することができます。 </li><li>固定の場合は、細胞を再懸濁し、混合するために各ウェルにピペットを4％パラホルムアルデヒド100μLを加える。 </li><li>ハイスループットフローサイトメトリー分析は、HTSプレートリーダーを搭載したBD LSR IIを使用して実行することができます。</li><li>プログラムソフトウェアは、各ウェルの3倍（100μlの混合量）を混合し、各サンプル30μlの、または少なくとも2000のセルのイベントを、分析する。 </li><li>収集されたデータは、FlowJoまたは同等のソフトウェアで解析することができます。有用なメトリックは、ビーズ+のパーセント、または蛍光細胞、現在の食細胞の数の尺度を提供するだけでなく、貪食ビーズの数の尺度を提供する食細胞の蛍光強度が含まれています。これらの値を乗算すると、統合された平均蛍光強度、またはiMFIを生成します。 </li><li>それぞれの食細胞によって貪食ビーズの平均数は1ビーズの平均蛍光によってiMFIを割ることによって計算することができます。 </li></ol> 7。代表的な結果影響を受けると影響を受けない被験者からの抗体サンプルの明確な差別があるはずです。図1Aは、HIVネガから抗体サンプルのFACSヒストグラムを提示tive（黒のトレース）とHIVポジティブ（グレーのトレース）の対象、および抗原特異的抗体の存在によって駆動増加食作用を示しています。 アッセイの最適な感度はビオチン化抗原とビーズの飽和に依存しています。図1Bは、抗原の異なる量でコーティングされたビーズが（非結合抗体、三角形含む）3コントロールモノクローナル抗体でオプソニンされたときに観測された貪食を提示し、この抗原に対して飽和濃度として2μg抗原/μLビーズを確立します。 用量 - 反応曲線を図2に提示され、0.05から5μg/ mlの抗体の濃度範囲で貪食を誘導するために対象抗体サンプルの微分容量を示しています。この差の貪食は、IgGサブクラスとグリコシル化状態などの力価又はFcドメインの特性の違いのどちらかによって駆動されることがあります。 THP - 1細胞​​は、私している場合magedは、蛍光顕微鏡により、ビーズ貪食の明確な証拠がある。図3は、抗体の非存在下で貪食取り込みの欠如を示す抗体オプソニン化（緑）と非オプソニン化（赤）ビーズとのインキュベーション後のTHP - 1細胞​​、の二つ63x静止画像を提示。タイムラプス顕微鏡が実行されると、蛍光ビーズの抗体特異的貪食取り込みは（動画1、20倍の倍率）はさらに顕著です。 前の仕事は、セルに関連付けられているビーズの内在化を認識しています、と主単球を用いた実験では、このハイスループットアッセイ（データは示さず）に貪食スコア（iMFI値）とよく一致している。 <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="図1"/> 図1。アッセイの品質管理。 1A、HIV陰性対象（黒のトレース）とHIV陽性被験者（グレーのトレース）から抗体のサンプルの貪食のフローサイトメトリーのヒストグラム。1B：サンプルの抗原のための最適なビードコーティング条件の実験的決定。抗原特異的なモノクローナル抗体（円、正方形）はビーズの&gt; 2μgの抗原/μlでコーティングしたビーズの最大の食作用を示し、対照抗体ながら、（三角形）は貪食能を示していない。 <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="図2"/> 図2。貪食用量-反応曲線。差HIVポジティブ（処理、未処理、および抗レトロウイルス療法の不在下でウイルス複製の制御を示す）とgp120のHIV陰性の被験者のドライブの貪食（HIVのエンベロープ）をコーティングしたビーズから臨床抗体サンプル。 <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="図3"/> 図3。効率的な内部化。非オプソニン化ビーズは、ゾル中に残したまま顕微鏡では、抗体-オプソニン化ビーズ（緑）の内在化を確認ution（63x倍率）。 映画は1。抗体主導貪食のタイムラプス顕微鏡は、14時間かけて行い、ハイスループットアッセイで使用するTHP - 1細胞の貪食能の可視化を可能にした。緑色蛍光ビーズは抗体オプソニンである、赤色蛍光ビーズは、負の制御を提供します。 <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">映画を鑑賞するためには、ここをクリック。</a>

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ここに記載のアッセイは、長い貪食のプロセス10とプレートベースの自動フローサイトメトリーを研究するために利用さ単球細胞株を利用することにより、臨床抗体サンプルの貪食能のハイスループット分析を可能にします。このアッセイではないだけ別の科目から、疾患特異的抗体によって駆動される食作用の違いの研究を可能にする、正確に抗原特異的抗体のサブセットを特徴づ​​けるために、その能力で他の人よりも有利ですが、また、同じサブジェクトに複数の抗原特異性の研究。単球や他の抗原提示細胞を含む生来のエフェクター細胞の抗体の採用が強く疾患の転帰11-13影響、およびFcドメイン14から16のAsparagine297で抗体の幾何学、IgGサブクラス、およびグリコシル化に応じて生物学的に可変なので、この方法はのために用意されていますアクションの重要な抗体機構の評価。さらに、抗体 - メディためated貪食が感染7,17,18の過程でいくつかの病原体によって悪用され、このアッセイは抗体依存性向上のプロセスの評価の可能性を秘めています、そして潜在的に保護だけでなく、潜在的に有害として食作用の二面性を強調抗体活性。 記載のアッセイは、患者集団の、だけでなく、ワクチン接種の臨床サンプルから抗体を分析するために利用することができる、という精製IgGより血清利用するために適合させることができる。さらに、食作用への応答におけるサイトカインの分泌は、培養上清から分析することができます、そして特定のFcRの影響は、FCR -ブロッキング抗体を用いて決定することができる、洞察力と決定される貪食能にだけではないの違いが、下流のシグナル伝達事象を、可能機構に、そしてこの免疫機構の理解を解決し、精度を高めることができます。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> EZ -リンクマイクロスルホ- NHS - LC -ビオチン化キット</td><td>サーモScientfic </td><td> 21935 </td><td></td></tr><tr><td> Ultracel - 50膜によるアミコンウルトラ- 15遠心フィルターユニット</td><td>ミリポア</td><td> UFC905024 </td><td>フィルターユニットのサイズは、抗原のサイズによって異なります</td></tr><tr><td> FluoSpheresニュートラビジンは、ミクロスフェア、1.0μmの、黄緑色の蛍光（515分の505を）というラベル</td><td>インビトロジェン</td><td> F - 8776 </td><td>メーカーは様々な色で本製品を生み出す</td></tr><tr><td>メロンゲルのIgG精製キット</td><td>サーモ</td><td> 45212 </td><td>ケアは、SDS - PAGEによる抗体試料の純度を検証するために注意が必要です。 </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

抗体主導貪食は、専門の食細胞上のFc受容体の関与を介して誘導され、クリアランスだけでなく、病気の病理学の両方に貢献することができます。抗体の可変ドメインのプロパティが長く生体機能のために重要と考えられているが、それらのFcドメインを介して自然免疫細胞を募集する抗体の能力は、両方の組換えの設定で、その有効性の主要な要因としてますます感謝になっているだけでなく、モノクローナル抗体療法、自然感染またはワクチン接種1-3の経過インチ重要なのは、一定のドメインとして、その命名にもかかわらず、抗体のFcドメインは、一定の機能を有していない、およびIgGサブクラス（IgG1の- 4）とアスパラギン297 4-6のグリコシル化によって強く変調される。したがって、臨床サンプル中の抗原特異的抗体の機能の違いを研究するためには、このメソッドは、貪食ポットの相関関係を容易にする病気の状態、感染症への感受性、進行、または臨床転帰に対する抗体の動。 さらに、このエフェクター機能は、Fc受容体主導の貪食7を介して宿主細胞に病原体へのアクセスを提供することで感染を強化するために抗体の文書化能力の光の中で特に重要です。また、免疫複合体の貪食取り込みが免疫応答8のTh1/Th2分極に影響を与えることができるいくつかの証拠がある。 ここでは、差動IgGサブクラス、Asn297における糖鎖構造だけでなく、ハイスループットな方法で抗原特異的抗体の免疫複合体を形成する能力によって発生する可能性がある抗体誘発性食作用の違いを、検出するように設計されたアッセイを説明。この目的のために、1μmの蛍光ビーズを蛍光抗原特異的免疫複合体を生成し、臨床抗体サンプルとインキュベート、抗原でコーティングされています。これらantiboDY -オプソニン化ビーズを抑制し、活性化の両方を含む複数のFcγRsを発現する単球細胞株とインキュベートされる。アッセイの出力は、貪食能、サイトカイン分泌、およびFcγRsの使用状況のパターンを含めることができ、この感染症とワクチンを介する保護9の両方でこの抗体依存性エフェクター機能の解析の違いのため非常に有用なシステムとなって、標準化された方法で決定される。

Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

Determining the Phagocytic Activity of Clinical Antibody Samples

我々は、蛍光抗原をコーティングしたビーズと標準化で複数のFc受容体は、提供する受容体の使用量と貪食能の測定を表す単球細胞株を利用し、臨床検体から抗原特異的抗体の食作用活性を決定するためにハイスループットフローサイトメトリーアッセイを提示し、興味のある任意の抗原のための再現可能なファッション。

臨床抗体サンプルの貪食活性を決定する

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

determining-the-phagocytic-activity-of-clinical-antibody-samples

Research

JoVE Journal

Immunology and Infection

23.4K ビュー.  Ragon Institute of MGH, MIT, and Harvard. 我々は、蛍光抗原をコーティングしたビーズと標準化で複数のFc受容体は、提供する受容体の使用量と貪食能の測定を表す単球細胞株を利用し、臨床検体から抗原特異的抗体の食作用活性を決定するためにハイスループットフローサイトメトリーアッセイを提示し、興味のある任意の抗原のための再現可能なファッション。

ビデオ: Determining the Phagocytic Activity of Clinical Antibody Samples

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

赤血球凝集アッセイを用いて血清の反応性と力価の決定

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

免疫・感染学

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

ウイルス性病原体のために臨床サンプルをスクリーニングするためにパンウイルスマイクロアレイアッセイ（Virochip）を使用して、

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

オリゴヌクレオチド結合蛍光マイクロスフェアを使用して複雑な臨床及び環境試料中の細菌の多重検出

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

xCELLigenceシステムにCR51リリースアッセイを比較することによって、人間Her2neu特異的CD8 T細胞の最適な細胞傷害活性を決定する

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

哺乳動物細胞に関連して細菌の生存を決定するための蛍光顕微鏡法

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

ファージディスプレイされた合成抗体ライブラリーから、スケーラブルな高スループットの選択

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

用いた環境サンプルから新規Faustovirusesの単離のための迅速な戦略<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

白血球に富んだ血液サンプル中のハロゲンペルオキシダーゼ活性の迅速かつ具体的な評価

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...