MRSへの補助金によって部分的なサポートはカナダ自然科学工学研究評議会（NSERC）、神経科学研究のためのハリーBotterell財団、ARC、およびガーフィールドケリー循環器の研究開発基金からのものです。 SRAは、クイーンズ大学の研究賞とロバートJ.ウィルソンフェローシップの受賞者です。 PJは、女王の大学院賞、ロバートJ.ウィルソンフェローシップの受賞者です。 AGとSAは、女王の大学院賞の受賞者です。 SFは科学技術（OGSST）のオンタリオ大学院奨学金の受け手だった。 AGは、女王のフランクリンブラッケン大学院奨学金の受取人です。 SAは、女王のRS McLaughlinは大学院奨学金の受取人です。

1。復活冷凍マクロファージ細胞<ol><li> -80℃冷凍庫から凍結細胞を復活させる前に、人は10％ウシ胎児血清（FBS）とplasmocin 5μg/ mLのを補充した滅菌濾過したダルベッコ変法イーグル培地（DMEM）を用いて培養液を準備する必要があります。 Plasmocinは、細胞培養のマイコプラズマ汚染を排除し、防止するために私たちの研究で使用される抗生物質溶液です。 </li><li>凍結細胞の1バイアルの場合、一つの4mL、無菌のバイオハザード封じ込めフードで25cm 2の細胞培養フラスコに培養液1 mLを追加します。 </li><li>凍結細胞のバイアルは-80℃の冷凍庫から取られている場合、それらはすぐに約2〜3分かかる滅菌フード、で手で温めることによって解凍されています。 </li><li>とすぐに細胞が融解しているとして、彼らはすぐに1mLの滅菌ピペットを用いて馴化培地を含むフラスコに転送されます。 </li><li>細胞を含むフラスコを37℃、5％CO 2で組織培養インキュベーター内に配置されています。 </li><li>彼らは倒立顕微鏡を用いて75〜90％コンフルエントになるまで細胞が日常的に成長し、遵守をチェック。 </li><li>細胞培養のインキュベーションの間に、12 mm径の円形のガラスのスライドがバイオハザード封じ込めのキャビネットで1時間に大きなガラスのペトリ皿に70％エタノールでそれらをカバーすることによって滅菌され、アルコールが、後で削除しreusageのために保管し、ウェットガラスの両側れで24時間またはそれらが乾燥しているまで、UV下キャビネットの滅菌濾過空気の流れにさらさ残されています。 </li></ol> 2。シアリダーゼのアッセイのためのめっきセル<ol><li>細胞を播種する前に、一つの滅菌円形ガラススライドは慎重にBDファルコンの1つのウェルで24ウェル平底滅菌炎完全な曲線を使って蓋組織培養処理されたポリスチレンプレート、4&quot;、鋸歯状、ステンレス鋼forcepで配置され。75％コンフルエントの細胞の1つのフラスコでは、通常、培養プレートの12ウェルを使用してください。 </li><li>接着マクロファージ細胞の場合は、DMEM条件培地を滅菌封じ込めのキャビネットに5 mLの滅菌ピペットを用いてフラスコから削除されます。任意の血清含有培地を除去し、カルシウム培地フリー（CMF）リン酸1 mLを追加するには、生理食塩水pH7.4をバッファ1X滅菌トリスで一度付着細胞を洗浄し、細胞をカバーするのに十分なpH7.4の緩衝食塩水。細胞がフラスコで離陸を開始するまで、細胞は約10分間または37℃のインキュベータ内に配置されています。フラスコを軽く揺り動かしたり、残りの接着細胞を緩めるために振とうすることができます。 </li><li>フラスコ内の細胞懸濁液を1 mLに、DMEMの条件培地3 mLを加える。細胞懸濁液0.5 mLをとり、滅菌12ミリメートルの円形のガラスのスライドを含む組織培養プレートにそれらを転送する。約、0.5 × 10 6細胞は円形のガラスのスライドに転送されます。 </li><li>細胞を含む組織培養プレートは、細胞が円形のスライドガラスに付着することができるようにするために、37℃で24時間、5％CO 2のCでインキュベートする。 </li></ol> 3。シアリダーゼアッセイ 1。コントロールの作成<ol><li>最後に、4 MUNANAの1μLDAKO + 1μLと+：顕微鏡スライド（つや消し：1終了、1slide、25 × 75 × 1mm）の置いて封じ込め、キャビネットまたはラボのベンチでをと順番に混合することにより、以下の化合物を追加します。トリスの3μLは、生理食塩水pH7.4の（TBS）をバッファ。 </li><li>よく細胞を含むいずれかからメディアを削除した後、優しくものコーナー〜4&quot;forcepの円形ガラススライドを持ち上げます。慎重forcepでガラススライドを削除し、上の混合溶液に直面している細胞をスライドに配置顕微鏡のスライド。 </li><li>すぐに、UVフィルタに設定されていると蛍光画像をキャプチャするためのデジタルカメラを持っている落射蛍光顕微鏡にmicrocopeのスライドを取る。 </li><li>もし蛍光灯モードに切り替えてセルを参照してできるようになるまで位相コントラストの下顕微鏡フォーカス。 1分、2分、3分で蛍光灯の下での細胞の写真を撮る。位相コントラスト下で細胞の単一の写真を撮る。 </li></ol> 2。正のテストを行って<ol><li> LPSの4 MUNANA + 2μL+ TBSの1μLの1μLDAKO + 1μL：別の顕微鏡スライド上に順番に以下の化合物を見つける。 </li><li>上記の手順3.1.2に3.1.4を繰り返します。 </li></ol> 3。ノイラミニダーゼ阻害剤タミフルと一緒に正のテストを作る<ol><li> 1μLDAKO +1μLLPSの4 MUNANA + 2μL+タミフルの1μLの：別の顕微鏡スライド上で、次の順番で以下の化合物を見つける。 </li><li>円形のガラススライド上に生きている細胞と接触する化合物の最終濃度が5の希釈係数を持つ、タミフルの濃度は、生理食塩水pH7.4をバッファ1Xトリスにあらかじめ用意されています。 </li><li>上記の手順3.1.2に3.1.4を繰り返します。 </li></ol> 4。シアリダーゼ活性を50％抑制するのに必要な阻害剤の濃度の決定（IC 50） <ol><li>細胞を取り巻く蛍光を定量するために、画像がRBGを測定するための分析プラグインでImageJを1.38xを使用して分析している。細胞を取り巻く蛍光の50ランダムスポット測定値を取ると、総蛍光の平均値を計算する。 </li><li> IC 50は、非線形回帰を用いてy軸上の平均の蛍光に対してx軸上（例えば、ログng / mL）の対数単位として変換阻害剤の濃度のプロットから生成されます。 </li></ol> 5。成功の秘訣<ol><li>培養中の細胞がマイコプラズマのコンタミであることを確認してください。我々は日常的にこれを制御するための培養液中でPlasmocinを使用してください。 </li><li>人工的なノイラミニダーゼ基質、4 MUNANAは、作りたての使用する必要があります。調製した基質は、シアリダーゼのアッセイのための週間以内に使用することができます。 </li><li>細胞株は、細胞表面上の低受容体の発現がある場合、人は刺激のための配位子の高用量を使用することができます。 </li><li>我々はまた、6回以上にわたって継代細胞はその受容体の表現型が失われる可能性があることを経験している。たての、復活の細胞を培養する必要があります。 </li></ol> 6。代表的な結果添付の代表的な結果とシアリダーゼアッセイのアニメーションプロトコルを参照してください。 <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">PowerPointファイルを</a> 。

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRAとPJは第一著者として平等に貢献した。

ライブマクロファージ細胞2でシアリダーゼ活性を検出するために新しく開発されたアッセイを使用して、我々としても、ライブDC - 2.4樹状細胞の用量依存的にライブBMC - 2マクロファージ細胞におけるリガンド誘発性シアリダーゼ活性のシアリダーゼ活性を検出するためにこの技術を使用し、HEK-TLR4/MD2、HEK293、SP1乳腺腺癌細胞、ヒトWTおよび1140F01とWG0544 I型sialidosis線維芽細胞。タミフ?...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
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<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
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<td>Tamiflu (Oseltamivir Phosphate)</td>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

哺乳類のToll様受容体（TLR）は病原体関連分子パターンを認識する受容体ファミリーです。だけでなく、TLRは微生物の重要なセンサー（例えば、ウイルス、細菌および寄生虫）感染症である、彼らはまた、おそらく自己免疫疾患で感染症、炎症性疾患、との病態生理に重要な役割を果たす。このように、感染症に対するTLR応答の強度と持続時間は厳密に管理する必要があります。それはセンサーの受容体の構造的完全性を理解すること、次の、そのリガンドの相互作用とシグナル伝達成分は、その後の免疫学的防御のために不可欠です。また、センサーの操作による疾患修飾のための重要な機会を提供するであろう。 TLRのセンサーのシグナル伝達経路はよく特徴付けされていますが、センサーとそのリガンドとの相互作用を制御するパラメータは、まだ明らかにされないままである。我々は最近、以前に1,2を観察されていない、その天然のリガンドによるTLRの活性化の新たなメカニズムを同定した。それは、リガンド誘導TLRの活性化が密接にNeu1シアリダーゼの活性化によって制御されていることを示唆している。我々はまたNeu1がしっかりNeu1とマトリックスメタロプロテイナーゼ-9（MMP - 9）受容体4との複合体におけるクロストークを含むであるTrkAとTrkB 3などのニューロトロフィン受容体に、調節していることを報告している。シアリダーゼのアッセイは、最初はGPCRGαiタンパク質およびMMP - 9の5を介してマクロファージ、樹状細胞と線維芽細胞の細胞表面上Neu4シアリダーゼの活性化で、小説リガンド、thymoquinoneを見つけるために使用されています。 TLR受容体のために、我々のデータはNeu1シアリダーゼはTLR - 2、-3および-4受容体との複合体に既に存在し、いずれかの受容体へのリガンドの結合によって誘導されることを示している。 TLR - 4量体化、MyD88/TLR4複​​雑な募集、NFkBの活性化​​および炎症誘発性細胞応答に立体障害を除去するために結合するリガンドから遠いNeu1シアリダーゼ加水分解シアリルα- 2、3 - リンクされているβ-ガラクトシル残基を活性化。共同報告書では、Neu1シアリダーゼはマクロファージの細胞6食作用を調節することが示されている。一緒になって、シアリダーゼのアッセイは、リガンド誘導性受容体の活性化の分子メカニズムへの強力な洞察を提供してくれました。 Neu1シアリダーゼとTLRの活性化との間の正確な関係は、TRK受容体がまだ完全に解明されているものの、それは細胞調節経路に新しいまたは先駆的なアプローチを表すことになります。

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

シアリダーゼのアッセイは、微生物感染と生マクロファージの細胞表面の受容体レベルでの炎症性疾患への関与のTLRセンサの新しい分子機構（s）を解明する簡単な技術的なアプローチです。

Toll様受容体の活性化を調節するのNeu1シアリダーゼ活性の検出

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K ビュー.  Queen's University - Kingston, Ontario. シアリダーゼのアッセイは、微生物感染と生マクロファージの細胞表面の受容体レベルでの炎症性疾患への関与のTLRセンサの新しい分子機構（s）を解明する簡単な技術的なアプローチです。

ビデオ: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

薬物過敏症におけるIgEが介在する機序の解明のための好塩基球の活性化試験

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

ヒト血液の小さなサンプルから単球および樹状細胞のlineage特異的Toll様受容体を介するシグナル伝達の定量的イメージング

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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 ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

赤血球上の抗体誘導性補体活性化の定量的検出のための方法

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

ヒト単球由来樹状細胞におけるIL-1βを用いて活性化し、NLRP3ソーム活性の測定

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

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. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

単離とマウスリンパ球の活性化

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.