SZとDWがViropharmaの無制限の助成金を受ける。

1。ブロメライン処理した赤血球の準備<ol><li> 1X PBS（2-5分間664×gの遠心分離）で3回0に型指定された赤血球を洗浄します。 </li><li> 37℃で10分間、0.5％ブロメライン溶液を2倍量のパックされたRBCの1の体積をインキュベート</li><li>細胞は1×PBSで3回洗浄します。 </li><li>細胞は、少なくとも1週間1×PBS中で4℃で3％の溶液として保存することができる。 </li></ol> 2。 FACSによる赤血球上のC3、C4の堆積分析<ol><li> 56℃で30分間サンプルを加熱することによって患者の血清（または他の抗RBC抗体源）の必要量を、熱不活性化</li><li>ブロメライン処理した赤血球のVBG 3回洗浄する。 - （5mMのベロナール、150mMのNaCl、0.05％ゼラチン液pH7.4）およびVBG + +（VBG - + 2mMのMgCl 2 + 10mMのCaCl 2）に再懸濁し得0.5％溶液。 </li><li>ガラスパール（fで丸底プレートには、次のコンポーネントをピペットまたは適切な混合）：25μlの0.5％赤血球、37.5μlの新鮮なAB血清（補体源）と37.5μL200μg/ mlの抗C5。抗C5は高価であり、入手が困難な場合があります。一つはC5-/C6-/C7-またはC8-欠損血清の代わりに、AB血清および抗C5の使用を検討して可能性があります。 </li><li> 0.1から33パーセントの最終濃度になるようにウェルあたり患者血清を追加します。正しい熱不活性化AB血清を用いによりウェル当たり血清濃度の違いのために。よく150μL/の最終容量を得るためにVBG + +を追加します。 ELISA箔との緊密なプレート。 </li><li>振とうしながら37℃で1.5〜2時間インキュベートする。 </li><li> PBS +0.5％BSA（2分間664×gの遠心分離）でRBCを3回洗浄する。 </li><li> 1μg/ mlの終濃度になるようPBS + 0.5％BSA中で（凝集を低減するために）蛍光標識した抗C3および抗C4モノクローナル抗体またはそれらのFabフラグメントを追加する。ここでは、カスタム抗C3-アレクサフルーア488を開発し、抗C4-アレクサフルーア647モノクローナル抗体が使用され、テサロニケの選択電子蛍光標識は、FACSによってC3及びC4の同時検出を可能にする。 </li><li>穏やかに振盪しながら、室温で30〜45分間インキュベートする。 </li><li> PBS +0.5％BSAで洗浄赤血球3X。 </li><li> PBS +0.5％BSA中に再懸濁赤血球150μLと（FACS分析の前にガラスパールを取り除くために）新しいプレートにそれらをピペット。 </li><li> FACS分析を行う。スキャッタプロットFSC-Aのダブレットとは別の単一細胞は、単一の赤血球上にゲートを設定し、蛍光シグナル（ 例えば 、FITCとAPC）のための適切な検出チャネルを選択します。 </li><li>蛍光強度中央値を使用して結果を定量化する。 </li></ol>注：これは、（ 例えば、蛍光標識抗IL6標識マウスIgG1）アイソタイプコントロールを使用することが可能である。しかしながら、それは、患者血清（AB血清のみ）なしで、または補体源（熱不活性化AB血清および熱不活性化患者血清）なしで赤血球をインキュベートすることによって、例えば、真の陰性対照を含むことが推奨されるDそして同じ抗C3-アレクサフルーア488および抗C4-アレクサフルーア647抗体でこれらを染色。これらの異なるコントロールは、典型的には、同じ、否定的な結果を与えるが、これは、赤血球12例におけるアイソタイプコントロールよりも多くの有効なコントロールを提供します。 3。定量的溶血アッセイ<ol><li> 56℃で30分間サンプルを加熱することによって患者の血清（または他の抗RBC抗体源）の必要量を、熱不活性化</li><li> VBGを洗浄赤血球3X -一度VBG + +と。 </li><li> （適切な混合用）ガラスパールと丸底プレートには、次のコンポーネントをピペット：35μlの3％赤血球、25μlの新鮮なAB血清（補体源）と1から50パーセントの患者の血清。熱不活性AB血清を用いて、血清濃度の差を補正。よく150μL/の最終容量を得るためにVBG + +を追加します。 ELISA箔との緊密なプレート。 </li><li>振とうしながら1.5〜2時間、37℃でインキュベートする。 </li><li> C言語減少減速（2〜3分で完全に停止するなど減速）で5分間664×gでentrifugeプレート。 </li><li>慎重に新しいマイクロタイタープレートに90μlの上清をピペット。なお、気泡を防止し、無傷の細胞沿っ取ることを防止するために、この段階で重要である。 </li><li>適切な分光光度計でA414/690を測定します。 </li><li>純水でサンプルRBCの溶解の割合として溶解を表現しています。 </li></ol>

<ol>
	<li>Zeerleder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+haemolytic+anaemia+-+a+practical+guide+to+cope+with+a+diagnostic+and+therapeutic+challenge.">Autoimmune haemolytic anaemia - a practical guide to cope with a diagnostic and therapeutic challenge.</a> Neth. J. Med. 69 (4), 177-184 (2011).</li><li>Reardon, J. E., Marques, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+and+transfusion+support+of+patients+with+autoimmune+hemolytic+anemia.">Laboratory evaluation and transfusion support of patients with autoimmune hemolytic anemia.</a> Am. J. Clin. Pathol. 125, 71-77 (2006).</li><li>Lai, M., Leone, G., Landolfi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hemolytic+anemia+with+gel-based+immunohematology+tests.">Autoimmune hemolytic anemia with gel-based immunohematology tests.</a> Am. J. Clin. Pathol. 139 (4), 457-463 (2013).</li><li>Zantek, N. D., Koepsell, S. A., Tharp Jr, ., R, D., Cohn, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+direct+antiglobulin+test:+A+critical+step+in+the+evaluation+of+hemolysis.">The direct antiglobulin test: A critical step in the evaluation of hemolysis.</a> Am. J. Hematol. 87 (7), 707-709 (2012).</li><li>Natukunda, B., Brand, A., Schonewille, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+alloimmunization+from+an+African+perspective.">Red blood cell alloimmunization from an African perspective.</a> Curr. Opin. Hematol. 17 (6), 565-570 (2010).</li><li>Packman, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+anemia+due+to+warm+autoantibodies.">Hemolytic anemia due to warm autoantibodies.</a> Blood Rev. 22 (1), 17-31 (2008).</li><li>Berentsen, S., Tj&#248;nnfjord, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+cold+agglutinin+mediated+autoimmune+hemolytic+anemia.">Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia.</a> Blood Rev. 26 (3), 107-115 (2012).</li><li>Petz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+antibody+autoimmune+hemolytic+anemia.">Cold antibody autoimmune hemolytic anemia.</a> Blood Rev. 22 (1), 1-15 (2008).</li><li>Barros, M. M. O., Blajchman, M. A., Bordin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warm+autoimmune+hemolytic+anemia:+recent+progress+in+understanding+the+immunobiology+and+the+treatment.">Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment.</a> Transfus. Med. Rev. 24 (3), 195-210 (2010).</li><li>Endoh, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+prewarming+conditions+for+Rh+antibody+testing.">Optimal prewarming conditions for Rh antibody testing.</a> Transfusion. 46 (9), 1521-1525 (2006).</li><li>Wouters, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C1-esterase+inhibitor+concentrate+rescues+erythrocytes+from+complement-mediated+destruction+in+autoimmune+hemolytic+anemia.">C1-esterase inhibitor concentrate rescues erythrocytes from complement-mediated destruction in autoimmune hemolytic anemia.</a> Blood. 121 (7), 1242-1244 (2013).</li><li>Arndt, P. A., Garratty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Review+of+Published+Methods+for+Analysis+of+Red+Cell+Antigen-Antibody+Reactions+by+Flow+Cytometry,+and+Approaches+for+Resolving+Problems+with+Red+Cell+Agglutination.">A Critical Review of Published Methods for Analysis of Red Cell Antigen-Antibody Reactions by Flow Cytometry, and Approaches for Resolving Problems with Red Cell Agglutination.</a> Transfus. Med. Rev. 24 (3), 172-194 (2010).</li></ol>

著者らは、開示することは何もありません。

上記のアッセイは、再現性と堅牢である。それらは、実施が容易であり、それは（96ウェルプレート）で同時に多数のサンプルを用いてそれらを行うことができる。したがって、このアプローチはまた、ELISAロボットシステム、例えば 、完全に自動化されたシステムに適しているであろう。現在使用される技術とは対照的に、これらのアッセイは定量的であり、これは、補体阻害剤の?...

赤血球（RBC）に対する抗体は、レシピエント赤血球上に存在しない抗原を発現する赤血球の輸血によって誘導することができる。これらのアロ抗体は、以下の輸血5時に補体活性化に起因する重篤な急性溶血性輸血反応を引き起こす可能性があります。自己免疫性溶血性貧血（AIHA）において、患者は、自分自身の赤血球に対する自己抗体を有する。これは、脾臓および/ ​​または肝臓6,7における補体受容体を介して補体を活性化することが自己抗体の場合には食細胞上のFcγ受容体と赤血球に結合したIgGの相互作用を介して細胞の加速クリアランスにつながる。血管内溶血をもたらし劇補体活性化は、稀ではあるが、多くの場合致命的である。加速、急性貧血ので、潜在的に致命的な組織低酸素のいずれかで同種もしくは自己抗体の結果によって誘導された媒介赤血球の破壊を補完します。 AIHAにおける自己抗体はdependin、暖かいと冷たい抗体に分類されている彼らは赤血球（それぞれ37℃以下）に結合し、最適な温度にグラム。暖かい抗体は、IgGアイソタイプおよびIgMアイソタイプ8,9の寒抗体の通常です。 AIHAは例えば lymphoprolyferative疾患、結合組織疾患、固形腫瘍、感染症や薬物への二次的であり得るが、症例の50％においてAIHAは特発9です。 γグロブリン（ 例えば、。のIgGまたはIgM）の検出、患者の赤血球に結合した補体は、半定量的な直接抗グロブリン（クームス）試験（DAT）によって行われる。 DATにおいて、患者RBCは抗IgGまたは抗C3dのとインキュベートする。 RBC凝集の発生が取り付けられ、補体成分の存在を実証またはIgG結合。同種または患者の血清中の自己抗体の検出は、間接抗グロブリン試験（IAT）を用いて行われる。 IATでは、ブロメライン処理した試験RBCは、患者の血清とインキュベートし、洗浄した後、抗HとインキュベートするIgGのUMAN。ケース赤血球に凝集が発生し、患者血清中の抗赤血球に存在するIgGで感作されています。赤血球に対するIgM抗体は、直接患者血清とブロメライン処理した試験RBCのインキュベーションで凝集につながる。直接クームス試験やIATにおける赤血球凝集反応を視覚的に試験管内の目によって、またはサイズ1によって凝集し、単一RBCを分離する小さなセファデックスカラムにサンプルをロードすることによって、どちらかに評価される。 赤血球上の補体活性化を測定するために、別の頻繁に使用される技術は、ブロメライン処理したRBC 10の（補体媒介）溶血を誘発する患者血清の容量が評価された溶血アッセイ1、である。遠心分離後の上清が原因で放出されたヘモグロビンを赤色に染色し、無色のままである場合は負であると考えられる場合に試験が陽性として注目されている。抗グロブリン試験や溶血アッセイの両方が最高出せるので、半定量的であるMの希釈はテストがまだ正である示されている。 抗グロブリン試験や溶血アッセイは、日常の診断に使用されている堅牢なアッセイである。これらのアッセイは、半定量的および補体阻害剤の有効性を評価する際に、必要に応じて、彼らは、赤血球上の補体活性化の微妙な違いを研究するのに適していないアッセイを行う技術者の経験に依存しているので。したがって、我々は我々がこの論文で説明赤血球に（オート）抗体による補体活性化を決定するために、2の定量的アッセイを開発した。 まず、赤血球上の補体C3およびC4の活性化フラグメントの沈着（ 図1A）を測定するためのアッセイを開発した。このアッセイにおいて、ヒトブロメライン処理したタイプ0 RBCは、熱不活性化患者血清（抗RBC抗体源）、新鮮なAB血清（補体源）および抗C5モノクローナル抗体（エクリズマブ）と共にインキュベートする。この社の間に患者血清は抗RBC抗体を活性化補完が含まれている場合ubation、C3、C4の沈着が発生します。下流の補体活性化によりRBC溶解を防ぐために、ブロッキング抗C5モノクローナル抗体が添加される。次に、赤血球上のC3およびC4沈着は、それぞれC3およびC4と反応する蛍光標識されたモノクローナル抗体またはFabフラグメントを用いてFACSによって検出される。単一の赤血球上のゲーティングは、結果の信頼性を確保することが重要である。この技術の利点は、カスケードの初期段階で、補体活性化、患者材料の小さな体積が必要であることを含んで評価し、この方法は、再現可能かつ定量的である。 C3およびC4の両方を見てのさらなる利点は、区別は、古典経路およびレクチン経路の活性化（C3とC4の沈着の両方）および代替経路の活性化（C3のみ堆積）の間に行うことができることである。代わりに、未処理のRBCのブロメライン処理したRBCの使用は、感度を増加させると言う。 第二のアッセイは、現在使用されて溶血アッセイ（ 図1B）に基づいている。ヒトブロメライン処理したタイプ0 RBCは、熱不活性化患者血清（抗RBC抗体源）と新鮮なAB血清（補体源）と共にインキュベートする。補数を患者抗RBC抗体によって活性化されている場合は、用量依存性のRBC溶解は、ヘモグロビンの放出につながるが発生します。放出されたヘモグロビンの量は、無傷の断片化RBCをスピンダウンした後、上清中の414 nmでの吸光度を測定することによって定量化される。吸光度が発生した溶血の量と相関する。現在使用されるアッセイとは対照的に、このプロトコルは、客観的な、非常に再現性およびアッセイの解釈者に依存しない溶血の定量値を可能にします。 これらの方法の適用はAIHAにおける補体阻害剤としてのC1-阻害剤の使用の可能性は11秒であった、に記載されているtudied。

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:195px;">Fresenius Kabi</td>
			<td style="width:119px;"></td>
			<td style="width:316px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B-4287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0261</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0263</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0470.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0238.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 488 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">46402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 650 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">62265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#945;C5 (Eculizumab)</td>
			<td style="width:195px;">Alexion Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS (Canto)</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;"></td>
			<td>Any FACS can be used that has the appropiate lasers.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer (e.g. Multiskan spectrum)</td>
			<td style="width:195px;">Thermo Labsystems</td>
			<td style="width:119px;">1500-193</td>
			<td>Any spectrophotometer with the right wavelength range can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSDiva software v 6.1.2</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">643629</td>
			<td>Any compatible FACS analysis software can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bromelain</td>
			<td style="width:195px;">Sanquin</td>
			<td style="width:119px;">K1121</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for quantitative detection of antibody-induced complement activation on red blood cells

赤血球（RBC）に対する抗体は、活性化の肝臓における補受容体を介して加速されたクリアランス（血管外溶血）を生じるか、RBCの血管内溶解をもたらすを補完する可能性があります。補体活性化につながる赤血球抗原に対する同種抗体（ 例えば ABO）または自己抗体（自己免疫性溶血性貧血、AIHAに見られるように）潜在的に有害であるとすることができます-血管内溶解をもたらす場合は特に-致命的な1。現在、原因のRBC ON（自動） -抗体に補体活性化は、RBCまたはRBC溶解1-4を反映した非定量溶血アッセイによる補体沈着を反映したクームス試験を使用することにより、in vitroで評価される。しかしながら、補体阻害剤の有効性を評価するためには、定量的な技術を有することが必須である。ここでは、そのような2つの手法を説明します。まず、患者の血清中の抗体により誘導される赤血球上C3及びC4沈着を検出するためのアッセイは、プレsented。このために、FACS分析を蛍光標識した抗C3または抗C4抗体とともに使用される。次に、定量的溶血アッセイが記載されている。このアッセイでは、患者血清により誘導される補体媒介性溶血が放出されたヘモグロビンの分光光度検出を利用して測定される。これらのアッセイの両方が抗体誘導性の補体活性化の研究を容易にする、非常に再現性および定量的である。

図2Aは、赤血球のための代表的な散布図を示しています。 FSC-プロット（P1） - シングル赤血球に適しゲーティングはデFSC-Wで見ることができます。通常、RBCの約95％がこのP1のゲート（単セル）内に入るが、患者血清中の高い割合が使用される場合、これは、抗IgMのRBCは、患者血清中に存在する場合は特に、70〜80％に低下することができる。 C4沈着にAIHA患者...

Watch this Scientific Journal Video about Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells at JoVE.com

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

ここでは、赤血球に対する抗体により誘導される補体活性化を測定するための2つのアッセイを記載する。現在のアッセイ以上の主要な利点は、それらの定量的かつ簡単に解釈する性質である。

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 ...

methods-for-quantitative-detection-antibody-induced-complement

Research

JoVE Journal

Immunology and Infection

30.4K ビュー.  University of Amsterdam. ここでは、赤血球に対する抗体により誘導される補体活性化を測定するための2つのアッセイを記載する。現在のアッセイ以上の主要な利点は、それらの定量的かつ簡単に解釈する性質である。 

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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

補体の枯渇により特定の細胞集団の枯渇

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

免疫・感染学

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

マウス腹腔細胞の分離

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

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 ...

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 ...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

タンパク質調製物、動物の予防接種と中和の検出：遺伝子組換えRBDベースのSARSのワクチンのためのプロトコル

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

High-throughput Detection Method for Influenza Virus

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

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

インフルエンザウイルスのためのハイスループット検出方法

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

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 ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性的および定量的検出のためのアッセイおよびタンパク質の抗菌薬のキャラクタリゼーション

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...