著者らは、国防総省を通じて議会監督医学研究プログラム（＃W81XWH-10-1から0445）、国立衛生研究所（NIH R21をHL110056）、および米国心臓協会（＃11SDG4890030）を資金認める。

1。バッファおよびテストエージェントの調製と滅菌<ol><li> 150mMのNaCl緩衝液：ナノ純水500ml中に4.383グラムNaCl結晶を溶かす。 </li><li> pH緩衝液：一塩基と二塩基性リン酸ナトリウムの適切な量を混合することにより、pHが5.6、6.2、6.8、および7.4のリン酸緩衝液を準備します。サンプルは低いpH値（ すなわち pHが&lt;5.6）などのクエン酸緩衝液として、より適切な緩衝液、でテストする場合は、使用すべきである。バッファのレシピは、容易に入手可能であり、例えば参照がここで提供されています12 </li><li>ボトルトップ吸引ろ過装置と再確認バッファーのpHを介して上記のようにすべてのバッファーを滅菌する。 </li><li> 20パーセントのTriton X-100（陽性対照）：ミックス20ミリリットル純粋トリトンX-100ナノ純水80mlインチ激しくボルテックスし、溶解するために超音波処理する。使用前に室温で一晩にしておきます。 </li></ol> 2。赤血球の調製<ol><li>血液frの25ミリリットルを得る凝固を防ぐために、K 2-EDTA-コーティングさバキュテイナー管に直接描かれたOMは、匿名の人間のドナー、。 </li></ol> 注：すべての手順は、適切な治験審査委員会（IRB）による事前承認を受けなければならず、静脈穿刺および採血がドナーへのリスクを最小限にするために訓練された瀉血専門医が行う必要があります。標準的な瀉血の手続きは、他の場所で公開されている13 <ol start="2"><li> 5分間、チューブ上のヘマトクリット値（赤、下層）と血漿（黄色がかった、上層）のマーク·レベルについては、500×gで血液を遠心分離します。 </li><li>マイクロピペッターを介して緩やかにプラズマを吸引除去し、漂白剤に追加して、バイオハザード廃棄物に捨ててください。 </li><li> 150mMのNaCl溶液でマークされた行（プラズマの元のレベル）にヘマトクリット管を埋める。キャップ、穏やかに混合するために数回転倒。 5分間、500×gで遠心分離します。 </li><li>再び血液細胞を洗浄するステップ2.3から2.4を繰り返します。その後supernatを吸引アリとpH7.4のPBSで置き換える。混在させる反転。 </li><li>テストされる各pHに対応する4つのチューブ内に均等に血を分割します。 （5.6、6.2、6.8、7.4）でテストする各pHに応じてチューブにラベルする。 </li><li> 5分間、500×gで採血管を遠心する。チューブ上のレベルにマークを付け、その後、上清を吸引除去する。 </li><li>適切なpH（2.6に示されるように）のバッファでマークされた行に各管を埋める。 </li><li>ラベル4 50 mlコニカルチューブ（試験へのpH当たり1）、および各コニカルチューブに適切なpHのPBSのピペット49ミリリットル。 </li><li> 1:50希釈のための対応するチューブに赤血球の1ミリリットル（同じpH）を追加します。視覚的に混濁されるべきであり、邪魔されずに放置しておくと落ち着きます希釈血液を検査。ペレットの形していない場合は、細胞を溶解した。 </li></ol> 3。溶解アッセイ96ウェルプレートのセットアップと定量<ol><li>テストする20X所望の最終濃度で、すべての実験薬物送達剤のストック溶液を準備します（アッセイ薬物送達剤+ 190μlの希釈赤血球、最終テスト混合物の中に、元の薬物送達剤の1/20希釈につながる）の10μlがかかります。 20、100、800μg/ mlの株式がそれぞれ1,5、および40μg/ mlのの最終試験濃度で、その結果、提案されている。 </li><li> V底96ウェルプレートに各ストック溶液の10μlのピペット。最適な結果を得るために、三重または四重に各サンプルをロードします。 </li></ol> 注：簡単にするために、それは独立した96ウェルプレートは、n = 3-4あたりのプレートにロードされた各サンプル（各濃度における）で、テストする各pHのために準備することをお勧めします。 <ol start="3"><li>正の対照ウェルは、10μlの20％Triton X-100を追加します。 </li><li>陰性対照ウェルは、リン酸緩衝液10μlを加える。テストする同じpHでバッファを使用します。 </li><li>希釈された赤血球のピペットで190μL（2.10参照）を各ウェルに、残っていることを確認細胞原液を作る転送中に均質。ヒント：このタスクを簡素化するためにマルチチャンネルピペットを使用してください。 </li><li> 1時間（：オービタルシェーカーやロッカーを使用して、オプション）を37℃でプレートをインキュベートする。 </li><li> 500はペレット無傷赤血球にxgで5分間プレートを遠心します。注：遠心分離機からプレートを外して、次のステップにそれを輸送する際、取り扱いに注意し、細胞ペレットを妨害しないように注意する。 </li><li>マルチチャンネルピペットを使用し、クリア、平底96ウェルプレートに各ウェルから100μlの上清を移す。注：細胞ペレットを誤って任意のサンプル（s）の妨害された場合は、1つは、再遠心することができますプレート後、上澄みの転送を続行します。 </li><li>プレートリーダーを用いて上清の吸光度を測定します。波長の範囲は（ - 541 nmの400）を使用することができることに注意してください。 </li></ol> 注：異なるプレートリーダーが異なる飽和点と感度を持っている可能性がありますので、私のために波長の選択asurementsは、界面活性剤処理により誘導されるような実験サンプルから溶血データが確実に最大溶血に対して正規化することができるかどうかによって異なります。これは、ポジティブコントロールサンプルの吸光度の値を正確に測定する必要があります。 <ol start="10"><li> Microsoft Excelまたは類似のデータ解析ソフトウェアを使用して、各pH（ステップ3.4）用に設定され陰性対照試料からのバックグラウンド吸光度の測定値の平均を求める。そのpHで測定した他のすべての試料から、このバックグラウンドの吸光度の値を減算します。 </li><li>バックグラウンドを差し引いた後、陽性コントロールの平均吸光度洗剤で処理したサンプル（ステップ3.3）を見つける。次いで100％溶血を表すべきであるこの平均吸光度値にすべての実験データ点を正規化します。最後に、洗剤コントロールに対する相対的なだけでなく、個々に発生した％溶血を計算するために100％で各ウェルの値を乗算します。 </li></ol>

<ol>
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IRBの承認：ヒトを対象とする手順は、バンダービルト大学治験審査委員会（;プロトコル＃111251 IRB）により承認されている。特別な利害関係は宣言されません。

-初期エンドソームのpH 6.2 -後期エンドソームのpH 5.6 - pHは6.8、エンドソーム溶解機能のために設計されたpH応答性ポリマーまたは他の薬剤を迅速かつ効果的にエンドソーム（ 図1に遭遇したpH値で赤血球の溶解に基づいてスクリーニングすることができるリソソーム）14から17、pH依存性溶血は、生体高分子の治療法（ 例えば、ペプチドは、siRNA、ODNは、タンパク質）...

細胞内の多くの潜在的な影響力の大きい治療標的がありますが、薬剤の細胞内送達は、重要な課題となっています。頻繁に、薬剤、特に生物学的製剤は、エキソサイトーシスを介して細胞によって内在化および小胞に人身売買どちらのendo-リソソーム経路を介して、それらの内容の劣化につながること、またはセルの裏に運ばれますされています。後者の過程で1、内部のpHが小胞の酵素の活性の至適pHである、約5から6に酸性化されているリゾチームなど、この区画内の関数は、図2 最近では、材料の数は、特にの酸性化は、その貨物の細胞質送達を容易にするエンドソームを活用するように操作されている。このアプローチの一つの例では、コア生理学的pH（ すなわち 7.4）で両性イオンと電荷的に中性である合成、高分子ミセルのナノ粒子を使用しています。しかし、pH6.0で- 6.5、ポリマーがプロトン化になるとミセルのコアを不安定正味の正電荷を取得し、露出したポリマーセグメントと相互作用し、エンドソーム膜を破壊する。この活動は、彼らの細胞質ゾルのターゲットにアクセスできるように、ペプチドおよび核酸ベースの治療薬のエンドソーム脱出を促進することが示されている。膜障壁を崩壊エンドソーム脱出を媒介するために開発された方法の3,4他の例は、または&#39;融合&#39;ペプチドが含まれるリン脂質二重層の膜融合や過渡孔形成を媒介することができるタンパク質は、例えば、ポリ（プロピルアクリル酸）のようなアニオン性アルキルアクリル酸の5ホモは別のよく研究されたアプローチであり、これらのポリマーに、ペンダントカルボン酸のプロトン化状態が遷移を指示エンド-リソソームのpH範囲において、疎水性膜の中断状態になります。6,7 エンドソーム溶解挙動をスクリーニングするための一つの有用なモデル系はeですX生体内pH依存性溶血アッセイ。このモデルシステムでは8、赤血球膜は、エンド-リソソーム小胞を囲む脂質二重膜の代用として機能します。この一般化モデルは細胞透過性ペプチドおよび他の高分子遺伝子送達システムのエンドソーム溶解挙動を評価するために他の人によって使用されています8月11日今回の実験では、ヒト赤血球および試験材料は模倣する定義されたpHで緩衝液中で同時インキュベートアール、（7.4）は、細胞外（6.8）初期エンドソーム、およびエンドリソソーム後期（&lt;6.8）環境に対応しています。潜伏期間中に放出されたヘモグロビンの量は、洗剤を用いて溶解し、陽性対照試料で放出されたヘモグロビンの量に正規化されている赤血球の溶解、の尺度として定量化される。 潜在的にエンドソーム溶解試験材料の小さなライブラリーをスクリーニングすることから、1つは、pH7.4で全く溶血を生じないが、有意に上昇し、その裾のサンプルを推測することができますpHでolysis &lt;6.5は、細胞質ゾルの薬物送達のための最も効果的かつcytocompatible候補となります。これらの基準に適合する材料は、不活性のままで無差別にエンド-リソソーム区画に内在化後の局所pHの低下にさらされるまで、脂質二重膜（細胞毒性を引き起こす可能性がIE）を破壊しないことが予想される。 このプロトコルでは、赤血球はヒトのドナーから単離されており、実験的なエンドソーム溶解薬物送達剤を用いてpHを5.6、6.2、6.8、または7.4で共存培養した。無傷赤血球をペレット化し、上清を（溶解赤血球から放出されたヘモグロビンを含む）プレートリーダー（ 図1）を経由してヘモグロビンの吸光特性を分析する。

<table border="1"><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> BDバキュテイナー - K2EDTAバキュテイナー管</td><td>フィッシャー·サイエンティフィック</td><td> 22-253-145 </td><td>採血用</td></tr><tr><td> BDバキュテイナ採血針、20.5ゲージ</td><td>フィッシャー·サイエンティフィック</td><td> 02-665-31 </td><td>採血用</td></tr><tr><td> BDバキュテイナチューブホルダー/ニードルアダプタ</td><td>フィッシャー·サイエンティフィック</td><td> 22-289-953 </td><td>採血用</td></tr><tr><td> BDのブランドイソプロピルアルコール綿棒</td><td>フィッシャー·サイエンティフィック</td><td> 13-680-63 </td><td>採血用</td></tr><tr><td> BDバキュテイナーラテックスフリー止血帯</td><td>フィッシャー·サイエンティフィック</td><td> 02-657-6 </td><td>採血用</TD&gt; </tr><tr><td>塩酸（濃） </td><td>フィッシャー·サイエンティフィック</td><td> A144-500 </td><td> D-PBSのpHを調整するための。 </td></tr><tr><td>トリトンX-100 </td><td>シグマアルドリッチ</td><td> T8787 </td><td>陽性対照</td></tr><tr><td>ダルベッコPBS </td><td>インビトロジェン</td><td> 14190 </td><td></td></tr><tr><td>ナルゲンSFCAメンブレンMF75滅菌使い捨てボトルトップフィルターユニット</td><td>フィッシャー·サイエンティフィック</td><td> 09から740-44A </td><td></td></tr><tr><td> BDの96ウェルプレート平底、組織培養処理されたポリスチレン</td><td>フィッシャー·サイエンティフィック</td><td> 08から772-2C </td><td>アッセイの終了時に、板読書のため。 </td></tr><tr><td> BDの96ウェルプレート、丸底組織培養処理されたポリスチレン</td><td>フィッシャー·サイエンティフィック</td><td> 08-772-17 </td><td>実験的な薬剤と赤血球のインキュベーション。 </td></tr></テーブル&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

エンド-リソソーム小胞を構成するリン脂質二重層は細胞内標的に対する生物学的薬剤の送達に障壁をもたらすことができます。この障壁を克服するために、合成麻薬キャリアの数を積極的にエンドソーム膜を破壊し、細胞質内に貨物を提供するように設計されています。ここでは、細胞内薬物送達システムのcytocompatibilityとエンドソーム溶解活性のために迅速、ハイスループットスクリーニングとして使用することができ溶血アッセイを、説明します。 溶血アッセイ、ヒト赤血球および試験材料では、細胞外の早期エンドソーム、後期のendo-リソソーム環境を模倣する定義されたpHの緩衝液中で同時インキュベートされています。ペレット無傷赤血球に遠心分離工程に続いて、培地中に放出されたヘモグロビンの量を分光光度法（最高のダイナミックレンジの405 nm）で測定されます。パーセント赤血球の破壊は、その後陽性対照SAMPに対する相対定量化されLESは、洗剤を用いて溶解。このモデル系では赤血球膜には、エンド - リソソーム小胞を囲む脂質二重膜の代用として機能します。所望の結果は、生理学的pH（7.4）とpH約5から6.8からのendo-リソソームのpH範囲で堅牢な溶血で無視できる溶血です。

<html>典型的には、理想的なpH依存性溶血性挙動を示す薬剤は、薬物、核酸、または他の生物活性分子の細胞質送達のための最高の可能性を秘めている。これは、pH7.4で最小限の溶血を示し、 図2に描かエージェントとして第1に代表されるが、エンドソームのpH範囲（&lt;6.5）における溶血性行動が急激に増加しています。これらの薬剤は血液適合性ではない可能性があり、潜在的に?...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

溶血アッセイは、細胞内の貨物の配達のための薬物送達システム &quot;cytocompatibilityとエンドソーム溶解活性の急速な、高スループットの画面として使用することができます。アッセイは、環境pHの関数としての赤血球膜の破壊を測定します。

生体高分子薬の細胞質ゾルデリバリーのためのpH応答性エンドソーム溶解剤の評価のためにex vivoで赤血球溶血アッセイ

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

Research

JoVE Journal

Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

34.0K ビュー.  Vanderbilt University. 溶血アッセイは、細胞内の貨物の配達のための薬物送達システム "cytocompatibilityとエンドソーム溶解活性の急速な、高スループットの画面として使用することができます。アッセイは、環境pHの関数としての赤血球膜の破壊を測定します。

ビデオ: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokineticトラック運動アッセイによる細胞遊走の定量的評価

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

Bリンパ芽球様細胞株の樹立のためのシンプルな赤血球溶解法

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

変形能と赤血球による血液中赤血球の不均一性を測定

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...