プロジェクトはNIH NIAID契約番号HHSN2662000500021Cによってサポートされていました。我々は彼の技術支援のために明陳に感謝。

1。プログラムAmaxa 96ウェルシャトルNucleofector <ol><li>新しいパラメータファイルを開きます。 </li><li>あなたが96ウェルプレートの図の上にカーソルをドラッグすることによって、標準的なトランスフェクションに使用する井戸の数を選択します。各実験サンプルのプールに3ウェルの最小値を使用してください。 </li><li>入力プログラムコード：その1 SELECT&#39;FF&#39;のがpart2のselect &#39;168&quot;プルダウンメニューから</li><li>ソリューションボックスを選択し&quot;単球、人間の&quot;から</li><li>制御オプションの下に&quot;標準&quot;を選択します。 </li><li> Applyをクリックします。 </li><li>非トランスフェクションのコントロールを含めるには、コントロールのオプションから&quot;いいえプログラム制御&quot;を選択しないと適用をクリックし、必要に応じてダイアグラムからさらに井戸を選択し、。 </li><li>プレートダイアグラム上の任意の残りの未使用のウェルを選択して、定義解除をクリックしてください。 </li></ol> 2。トランスフェクションのためにDCを準備<ol><li>すべての作業が可能であれば、細胞培養フードに無菌条件下で行う必要があります。 450から2025までの比でヒト単球を96ウェルNucleofectorソリューションへの補足96ウェル添加することによりnucleofectionの溶液を調製します。混和後、室温まで昇温することができます。あなたは、ウェルあたりnucleofectionソリューションの20μlのが必要になります。エラーをピペッティングを可能にするために10％以上を行う。 </li><li>行1および2に最初のものを挿入すること、すなわち、正しい向きでnucleocuvetteプレートに必要なnucleocuvetteモジュールの数を置きます。 </li><li>あなたの実験は10分間400グラムで遠心分離することによっても、ペレットに50万​​セルに計算に必要なDCの数を決定します。注意深く上清を取り除く。 </li><li>軽く数回上下にピペッティングしてnucleofectionの溶液中で細胞を再懸濁します。 </li><li>特定の治療などGLOとRIG - I用のラベルeppendorfsに再懸濁した細胞の正しい量を割ります。 </li><li>ピペッティングで500,000個の細胞と混合あたり0.25μgのsiRNAを追加。あなたの非トランスフェクションのコントロールサンプルにおける非標的とするsiRNAを使用してください。 </li><li> nucleocuvetteモジュールに上記混合物のピペット20μlのは、あなたの実験的なレイアウトに応じて、液体を確保することは井戸の底に配信されます。 </li><li>気泡の除去を確保するために数回蓋付きnucleocuvetteプレートをカバーし、ハード面にプレートをタップします。 </li></ol> 3。トランスフェクションのDC <ol><li> Nucleofector 96ウェルシャトルのトレーにプレートを挿入し、アップロードをクリックしてから開始します。 </li><li>ラップトップのディスプレイ上にトランスフェクションのプロセスの進行に従ってください。赤い背景に黒いバーがそれが失敗したという意味のに対し、緑の背景に黒の十字架は、そのほかに成功したトランスフェクションを示します。 </li><li>トランスフェクションのプロセスの完了時に、プレートを取り外し、よくマルチチャンネルピペットを使用してそれぞれにDCの増殖培地の80μlを加える。 </li><li> 37℃および5％CO 2を 10分間インキュベートする。 </li><li>正しい方向を維持し、予め温めておいたDCの増殖培地100μlのを含む行列のチューブにnucleocuvettesから100μlのボリュームを転送する。 </li><li>トランスフェクションが発生していないそれらのチューブを取り外して廃棄。 </li><li> 37 ° C、5％の24時間またはその他の所望の時間間隔のためのCO 2。 </li></ol> 4。 NDVで細胞を感染させる<ol><li> eppendorfsに各実験サンプルのためにインキュベーターから細胞培養フードとプールのチューブに行列のチューブを取り外します</li><li>ペレット5分間卓上遠心機でスピンし、上清を除去することにより穏やかに細胞。 </li><li> MOI 1でNDVを含む無血清増殖培地中で細胞を再懸濁し、37℃と緩く無菌的に覆われてeppendorfs 5％45分間CO 2、。 </li><li> 80〜10時間還流DCの増殖培地の900μl、および再インキュベートする</li></ol> 5。細胞を回収<ol><li>卓上遠心機でeppendorfsを回転し、上清を除去することにより細胞をペレット化する。 </li><li>あなたのプロトコルに従ってRNAやタンパク質の抽出のための細胞を回収。 </li></ol> 6。代表的な結果： 私たちの最適化されたプロトコルを用いて、我々は、NDV（RIG - Iによって検出されたパラミクソウイルス）を持つ細胞はインターフェロン応答の経路を刺激するために感染して24時間とするためのリギ -標的とするsiRNAと樹状細胞をトランスフェクション。定量RT - PCR解析により、我々は、転写レベルで75％の遺伝子のノックダウンを示した。我々はまた、RIG - I IFNシグナル伝達カスケードの下流エフェクターであるIFNβの発現で同様の減少を、観察。さらに、MXA、IFNβ下流応答遺伝子、のそれは（図1A）最小限のだった一方、非感染、コントロール、トランスフェクトされた細胞におけるIFNβの発現は検出できなかったことを観察した。 と樹状細胞の第二のトランスフェクションリギ -標的とするsiRNAすることはウエスタンブロット解析を含むのに十分な細胞を用いて行った。ここで、RT - PCRの結果が（図1B）（62％、66％がノックダウンRIG - IとIFNβの発現をそれぞれ）我々が以前に見たものと似ていた、ウエスタンブロットはをプローブRIG - Iは、この遺伝子の発現があったことを明らかに完全に（図1C）がブロック。 <img src="/files/ftp_upload/2766/2766fig1.jpg" alt="図1A"/> 図1。 （A）MoDCsはRIG - Iまたは非特異的GLOのsiRNAと定量RT - PCRによって決定されるRIG - I、IFNβ及びMXAの発現に影響を標的どちらのsiRNAでトランスフェクションした。 （ - ）トランスフェクションプロトコルは、単球特有のバッファとnucleoporationプログラムFF168（ロンザウォーカー社）で24時間インキュベーションを使用する、トランスフェクションされた細胞は、いずれのNDV（+）または左に感染していないに感染させた。 10時間さらにインキュベーション後、細胞を回収し、RNA抽出された転写産物のレベルは、2つのレプリカの実験の結果を表していた。ボウルズら 16から撮影。（B）図1Aに使用されるように別のバフィーコートから得られたMoDCsが再度上記と同じトランスフェクションのプロトコルを使用して、いずれかのRIG - I -標的とするsiRNAまたは非特異的GLO siRNAでトランスフェクトした。トランスフェクトされていないMoDCsを使用して、追加のコントロールが実験に組み込まれた。 24時間のインキュベーションに続いてすべてのセルは、NDVを感染させ、RNAとタンパク質抽出の両方のために伐採される前に、さらに10時間インキュベートした。 RIG - I、IFNβ及び定量RT - PCRによって決定されるMXAの転写レベルは、2つのレプリカの実験の結果を表します。ボウルズら 16から撮影。（C）上記の図1Bに記載の細胞からのライセートをウェスタンブロットにより分析した。レーン1および2は、トランスフェクトされていない細胞からのライセート、レーン3および4は、GLOのsiRNAをトランスフェクションされた細胞からのライセート、レーン5および6は、RIG - I -標的とするsiRNAでトランスフェクトされた細胞からのライセート。サンプルは、RIG - IともGAPDH（ローディングコントロールとして）との重複で実行されたためにプローブした。ボウルズら 16から撮影。

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ナイーブな一次樹状細胞の効率的なトランスフェクションは、ハイスループット解析および自然免疫、獲得免疫の移行を仲介するこの重要な細胞内の細胞の炎症性経路のリバースエンジニアリングのために重要です。しかし、ほとんどの研究者はこれらの細胞は標準的なトランスフェクション技術を使用している場合の両方を効率的かつトランスフェクションの手順を誘導する細胞の成熟せ?...

<table border="1"><tbody><tr><th>機器/試薬</th><th>会社</th><th>カタログ＃ </th><th>コメント</th></tr><tr><td> Amaxa Nucleofector 96ウェルシャトル</td><td>ロンザ</td><td> 108S0109 </td><td>シリアル番号</td></tr><tr><td> Amaxaヒト単球の96ウェルNucleofectorキット</td><td>ロンザ</td><td> VHPA - 2007 </td><td>ヒト単球96ウェルNucleofectorソリューション、96ウェルサプリメントとNucleocuvettesとプレートが含まれています</td></tr><tr><td> RIG - I siRNAを</td><td> Dharmacon </td><td> L - 012511〜00 </td><td></td></tr><tr><td> GLOのsiRNAを</td><td> Dharmacon </td><td> D - 001600 - 01から20 </td><td></td></tr><tr><td> RPMI 1640 </td><td>インビトロジェン</td><td> 11875 </td><td> DCの増殖培地を作るために10％FCS、2mMのL -グルタミン、100 U / mlペニシリンおよび100μg/ mlのストレプトマイシンを補充した</td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 11965 </td><td></td></tr><tr><td> L -グルタミン</td><td>インビトロジェン</td><td> 25030081 </td><td></td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>インビトロジェン</td><td> 15070063 </td><td></td></tr><tr><td>ウシ胎児血清</td><td> HyClone </td><td> 3070.03 </td><td></td></tr><tr><td>樹状細胞</td><td>ニューヨーク血液センター</td><td></td><td> DCは標準的な手順を使用して、バフィーコートから精製されています</td></tr></tbody></table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

樹状細胞（DC）は、その開始及び感染1に応答において重要な役割を果たす免疫系の歩哨と見なすことができます。ナイーブなDCによる病原性抗原の検出は、病原体関連分子パターン（PAMPS）と呼ばれる特定の保存構造を認識することができるパターン認識受容体（PRRS）によるものです。 DCによるPAMPsの検出は、成熟樹状細胞への活性化と変革をもたらす細胞内シグナル伝達カスケードをトリガします。このプロセスは通常、他の炎症性サイトカインと一緒に1型インターフェロンの産生によって特徴づけられる、そのようなT細胞との相互作用が適応免疫応答2を開始する所属リンパ節への成熟DCのMHCIIとCD86および移行などの細胞表面マーカーのアップレギュレーション、 3。従って、樹状細胞は自然免疫と獲得免疫系をリンク。 様々な病原体へのDC応答の根底にある分子ネットワークを解明する能力は、これらのシグナル伝達経路の調節とその誘導性遺伝子のより良い理解に重要です。また、感染症や腫瘍に対するDCベースのワクチンの開発を容易に役立つはずです。しかし、研究のこのラインは厳しく、プライマリDCは4をトランスフェクトすることの難しさによって妨害されています。 このようなレンチウイルスシステムなどのウイルスの伝達方法は、、一般的に使用されていますが、このような複雑さやバイオ危険なリスク（関連するコストと）5,6,7,8のような多くの制限を運ぶ。また、ウイルス遺伝子産物の配信は、DC 9,10,11,12を形質導入、それらの免疫原性を増加させる。エレクトロポレーションは、混合結果13,14,15と一緒に使用、しかし、我々はハイスループットトランスフェクションのプロトコルの使用を報告し、決定的にその有用性を実証する最初のですされています。 本報告書では、限られた細胞毒性およびDCの成熟16の不在で、人間の一次樹状細胞のハイスループットトランスフェクションに最適化された商用プロトコルをまとめたものです。トランスフェクション効率（プラスミドGFPの）と細胞の生存率はそれぞれ50％以上、70％であった。定量RT - PCRは、IFNβのない上方制御を実証しないままFACS分析は、トランスフェクションされた細胞の成熟マーカーCD86とMHCIIの発現の増加の有無を設立。このエレクトロポレーションプロトコルを使用して、我々はsiRNAと標的遺伝子RIG - I、mRNAとタンパク質レベルの両方で重要なウイルス認識受容体16,17、ノックダウン効果を持つ樹状細胞の正常なトランスフェクションのための証拠を提供する。

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

我々は、細胞の成熟を引き起こすことなく、プラスミドDNAやsiRNAのどちらかで初代ヒト単球由来樹状細胞をトランスフェクトの効率的な方法として私たちの最適化されたハイスループットnucleofectionのプロトコルを提示する。我々はさらなる標的遺伝子のmRNAとタンパク質レベルの両方におけるRIG - Iの成功のsiRNAサイレンシングのための証拠を提供する。

細胞の成熟せずに樹状細胞の効率的なトランスフェクションに最適化されたプロトコル

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

optimized-protocol-for-efficient-transfection-dendritic-cells-without

Research

JoVE Journal

Immunology and Infection

20.2K ビュー.  Mount Sinai School of Medicine. 我々は、細胞の成熟を引き起こすことなく、プラスミドDNAやsiRNAのどちらかで初代ヒト単球由来樹状細胞をトランスフェクトの効率的な方法として私たちの最適化されたハイスループットnucleofectionのプロトコルを提示する。我々はさらなる標的遺伝子のmRNAとタンパク質レベルの両方におけるRIG - Iの成功のsiRNAサイレンシングのための証拠を提供する。

ビデオ: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

追跡調査のためのQdot ®二ナノとマウス骨髄由来樹状細胞の世代とラベリング

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

免疫・感染学

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

アレルギー性​​気道炎症時の肺樹状細胞の成熟および移行の解析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

効率的なためキラー人工抗原提示細胞（KaAPC）<em&gt;体外</em&gt;ヒト抗原特異的T細胞の枯渇

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

ヌクレオによるヒトTHP-1マクロファージの高効率トランスフェクション

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

代謝表現型が異なる未熟、熟女と寛容原性樹状細胞の生成

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

生理学的免疫原性樹状細胞を生成するための新規プロトコル

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

濃縮ヒト記憶B細胞集団からの所望特異性を持つ抗体の選択と回収のための単細胞スクリーニング法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

インビトロ転写mRNAによる一次マクロファージの高効率トランスフェクション

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...