マカク血とPBMCサンプルの博士PramodさんNehete夫妻バーティNehete、この作業は、部分的にNIHの助成金AI 46969からの資金によって支えられて、そしてすべての細胞培養培地は、中央メディア研究所で生産され、NIHの助成CAによって資金を供給され16672。

1）細胞調製と抗原刺激、2）表面マーカーの染色、3）細胞内サイトカイン染色、4）フローサイトメトリー：合計だけでなく、T細胞のメモリーのサブセットの詳細な表現型および機能解析のための手続は、4つの部分に分けることができます。分析。 1。細胞調製と抗原刺激<ol><li>両方たての分離だけでなく、凍結保存された末梢血単核細胞（PBMC）は、このプロトコルで使用されていました。 PBMCをFicoll -ハイパック（Histopaquen - 1077、Sigma - Aldrich社、セントルイス、MO）を用いて密度勾配沈降によるアカゲザルのヘパリンまたはクエン酸静脈血サンプルから単離された。 PBMCのアリコートを液体窒素で90％FCS、10％DMSO中で凍結保存した。 </li><li>低温保存されたPBMCを使用する場合は、冷凍PBMCのバイアルを液体窒素から取り出し、そして急速に穏やかに混合し、37℃の水浴中で解凍、凍結培地を除去するRPMI - 1640（HyClone研究所、ローガン、ユタ州）で洗浄し、完全なメディアに再懸[CM、10％を含むRPMI - 1640サプリメント熱不活化FCS（HyClone研究所）、2mMのL -グルタミン（Sigma - Aldrich社）、100U/mlペニシリン/ストレプトマイシン（Invitrogen社）、および6で培養 - ウェル組織培養プレート37℃で一晩加湿5％CO 2雰囲気でC。翌朝は、生存細胞数をトリパンブルーの色素排除法により決定し、CMに再懸濁した。 </li><li>細胞は、サイトカインの分析に続いて抗原特異的刺激に対する非特異的なために異なる方法で処理した：（a）ホルボール12 - ミリステート13 - アセテート（PMA）とイオノマイシン（I）（シグマ - アルドリッチ、セントルイスとの非特異的刺激に対してルイ、MO）、0.1ミリリットルの量で細胞のアリコート（1.0 × 10 6細胞/ウェル）を96ウェル組織培養プレート（BD Biosciences社、フランクリン湖、ニュージャージー州）の個々のウェルに播種した。 PMAとイオノマイシンは、それぞれ、50ng/mlおよび500ng/mlの最終濃度で使用されていました。 96ウェル組織培養プレートの最終的な総量は0.2ml/wellです：（b）の抗原特異的刺激の場合、容積1.0mlに1.0 × 10 6細胞を24ウェル組織培養プレート（BD Biosciences社の個々のウェルに播種した、井戸が以前に変更9で説明したように前処理したフランクリン湖、NJ）、。簡単に言えば、24ウェル組織培養プレートは4の50mmのTris溶液で2.5μg/ mlの/ウェルのヤギ抗マウスIgG（H + L）（キルケゴールとペリー研究所、ゲイサーズバーグ、MD）（PH 8.6）で被覆した° C一晩。プレートは滅菌PBSと共刺激抗体CD49dは、クローン9F10（BD Biosciences社、サンノゼ、カリフォルニア州）で2回洗浄した翌朝は、37℃で一時間インキュベートすることにより、10μg/ mlの時に追加/よく続いていた℃でインキュベーション後、プレートを室温で滅菌PBSで2回洗浄した。 six HIVエンベロープペプチド8のカクテルを含む、対象の抗原は、（各ペプチドの10μg/mlの最終濃度で添加した。単独でCMのセルを持つ追加の井戸をネガティブコントロールとして調製した。24各ウェルの最終的な合計容積ウェル組織培養プレートは1.0 mlであった。 </li><li>細胞は37℃、加湿5％CO 2雰囲気で、Cで6時間培養した。ブレフェルジンAは、（シグマアルドリッチ、セントルイス、MO）刺激の最終的な4.5時間のために10μg/mlで培地に添加した。その後、細胞を5 mlのポリプロピレンチューブに移し、冷たい（4℃）フローの洗浄緩衝液（ダルベッコPBS（DPBS、 カルシウム /マグネシウム2 -無料で洗浄し、2％熱不活性化と生活の技術を、ロックビル、MD）その後FCS）、および様々な蛍光標識抗体で染色するために処理。 </li></ol> 2。染色免疫蛍光表面マーカー<ol><li>刺激された細胞は、最初の4で1UL LIVE / DEAD修正可能なアクア蛍光反応性色素で染色した℃で30分間、暗所でC、コールドフローの洗浄緩衝液で1回洗浄。 </li><li>その後、次のように適切に滴定する蛍光標識モノクローナル抗体を細胞に添加した：抗CD3 PE - Cy7（SP34 - 2）、抗CD8 Alexa700（RPA - T8）、抗CD28 PerCP - cy5.5（L293） 、抗CD95 APC（DX2）と抗CD4パシフィックブルー（OKT4）、（サンディエゴ、カリフォルニア州）eBioscience社からCD4除くBD Biosciences社（フランクリン湖、NJ）からすべて、パシフィックブルー（OKT4）。細胞は暗所で4℃で30分間℃でインキュベートした。 </li><li>各実験のために報酬のコントロールと蛍光マイナス1（FMO）の両方は、10,11が利用されて制御します。補償のコントロールは、他の検出器に汚れ、個々の波及coeffecientsを決定するために利用された。補償制御の場合は、サルのいずれかから細胞懸濁液を含むチューブの完全なセットが個別に単一の色の染みのような蛍光コンジュゲート抗体で染色した。 FMOは、すべての試薬が関心のいずれかを含む多色染色の組み合わせであり、正の間の境界を決定するために使用されます自家蛍光レベルと完全に染色されたサンプル中に存在するデータを複製することで、ND負の人口。 </li><li>染色した細胞はその後、コールドフロー洗浄バッファーで洗浄し、固定/膜浸透溶液（BD Biosciences社、サンノゼ、カリフォルニア州）で修正された（この段階で細胞を4℃で一晩保存することができます° Cを暗所で）少なくとも10分間細胞内サイトカイン染色を行う前に。 </li></ol> 3。細胞内サイトカインの染色<ol><li>固定された細胞はフローの洗浄緩衝液で洗浄し、その後、紹介予定/（BD Biosciences社、サンノゼ、カリフォルニア州）洗浄バッファーを製造者の指示に従って追加されました。簡単に言えば、細胞は4℃で10分間1Xパーマ/ Wash Bufferを0.1ミリリットルでインキュベートした。·ダークのC。 </li><li>その後、細胞を4℃で60分間、適切に滴定し、FITC標識抗IFN -γ（B27）と抗IL - 2、PE標識（MQ1 - 17H12）とともにインキュベートした。·ダークのC。 </li><li>その後、細胞は細胞膜透過液でさらに2回洗浄し、 </li><li>最終洗浄後、細胞をDPBSで1％パラホルムアルデヒドで再懸濁し、24時間以内にフローサイトメトリー分析にかけた。 </li></ol> 4。フローサイトメトリー分析<ol><li>またはシアンADP（Dako社、カーピンテリア、カリフォルニア州）、染色した細胞はLSR IIフローサイトメーター（サンノゼ、カリフォルニア州BD Biosciences社）に買収された。 FACSデータがFlowJoソフトウェア（木スター、オレゴン州アッシュランド）を用いて分析した。 </li><li>図。 1は、代表的な動物とは異なるT細胞サブセットの解析に利用さゲーティングスキームを示しています。リンパ球は前方散乱光（FSC）対側方散乱（SSC）を介して第ゲートであり、その後のライブリンパ球は、SSCとアクア陰性人口に基づいて同定された。 T細胞はCD3 CD4の検出+ CD8 -（CD4 + T細胞）に続く発現とCD4 - CD8 +（CD8 + T細胞）によってその後CD3 + T細胞集団内の個体積極的に同定された。 CD4 +およびCD8 + T細胞は、さらにナイーブ（Tnは、CD28 + CD95 -）、中央のメモリ（TCM、CD28 + CD95 +）とエフェクターメモリー（TEM、CD28 - CD95 +）細胞と同様にCD28とCD95の発現に基づいて異なるサブセットに区別された文献5、10で説明しています。 </li><li>図。 CD4 +およびCD8 + T細胞の異なるサブセットがPMAとイオノマイシン（PMA + I）による刺激に応答サイトカイン産生の点で機能的能力（INF -γおよび/またはIL - 2）について評価されたかを図2に示す。 </li></ol> 5。代表的なFACSデータアカゲザルのPBMC組成物は、FACS分析を用いて決定した。図1は、代表的な動物とは異なるT細胞サブセットの解析に利用さゲーティングスキームを示しています。リンパ球は前方散乱光（FSC）対側方散乱（SSC）を介して第ゲートであり、その後のライブリンパ球は、SSCとアクア陰性人口に基づいて同定された。 T細胞は、その後、CD3の発現により同定した。 CD4 + CD8 -（CD4 + T細胞）とCD4 - CD8 +（CD8 + T細胞）はCD3 + T細胞集団内の個体数も測定した。図2に示すように、CD4 +およびCD8 + T細胞は、さらにナイーブ（Tnは、CD28 + CD95 -）、中央のメモリ（TCM、CD28 + CD95 +）とエフェクターメモリー（TEM、などのCD28とCD95の発現に基づいて異なるサブセットに区別されたCD28 - CD95 +）細胞。図2Aは、INF -γを示し、非刺激およびPMA /イオノマイシン刺激CD4 + T細胞、およびサブセット、および図2Bにおいて、IL - 2産生は、INF -γおよび非刺激およびPMA /イオノマイシンはCD8を刺激+ T細胞におけるIL - 2産生を示し、およびサブセット異なるサイトカインプロファイルは、各サブセットの刺激によって誘発さに注意してください。 <img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="図1" border="0" /> アカゲザルmacqauesのPBMC成分のフローサイトメトリー解析に使用する図1。ゲーティング戦略。してください<a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="図2a" border="0" /> <img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="図2b" border="0" /> 図2。 （A）総CD4 + T細胞とサブセット。のサイトカイン産生プロファイル合計CD8 + T細胞とサブセットの（B）サイトカイン産生プロファイル。してください<a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg">ここをクリックして</a>大きな2aのバージョン、見て<a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg">ここにまたはを</a>図2bの大きいバージョンのために。

<ol>
	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, 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=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

ソリューションおよびバッファ<ul><li>フィコール - ハイパック（Histopaquen - 1077、Sigma - Aldrich社、セントルイス、MO） </li><li> RPMI - 1640培地（HyClone研究所、ローガン、ユタ州） </li><li>ウシ胎児血清（FBS）、（HyClone研究所、ローガン、ユタ州） </li><li> L -グルタミン（Sigma - Aldrich社、セントルイス、MO） </li><li>ペニシリン/ストレプトマイシン（Invitrogen社、カールスバッド、カリフォルニア州） </li><li>トリパンブルー試薬（シグマアルドリッチ、セントルイス、MO） </li><li> 50mMのトリス、pH 8.6、4でフィルタリングし、店舗° C </li><li>ダルベッコPBS（DPBS、Ca2/Mg2-free、生活技術、ロックビル、MD） </li><li>フローの洗浄緩衝液：2％FBSとDPBSサプリメント、4℃で保存</li><li> BD Cytofix / Cytoperm固定/透過処理ソリューションキット（BD Biosciences社、サンノゼ、カリフォルニア州） </li></ul>活性化剤<ul><li>ホルボール12 - ミリステート13 - アセテート（PMA）（Sigma - Aldrich社、セントルイス、MO） </li><li>イオノマイシン（シグマアルドリッチ、セントルイス、MO） </li><li>共刺激抗体CD49dは、クローン9F10（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li>アフィニティー精製抗体のF（ab）ヤギ抗 - マウスIgG（H + L）の2フラグメント（キルケゴールとペリー研究所、ゲイサーズバーグ、MD） </li><li>特異的活性化剤は、ペプチドのプール、単一のペプチド、または蛋白質全体を含めることができます</li></ul>サイトカイン分泌の阻害<ul><li> Brefedin A（BFA、シグマアルドリッチ、セントルイス、MO） </li><li>ゴルジプラグは（;サンノゼ、カリフォルニア州BD Biosciences社）代わりに使用することができます。 </li></ul>抗体<ul><li> LIVE / DEAD修正可能なアクア蛍光染色キット（Invitrogen社、カールスバッド、カリフォルニア州） </li><li> CD3 PE - Cy7（SP34 - 2）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li> CD4パシフィックブルー（OKT4）、（eBiosciences（サンディエゴ、カリフォルニア州） </li><li> CD8 Alexa700（RPA - T8）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li> CD28 PerCP - cy5.5（L293）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li> CD95 APC（DX2）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li> IFN - FITC（B27）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li><li> IL - 2のPE（MQ1 - 17H12）、（BD Biosciences社、サンノゼ、カリフォルニア州） </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

アカゲザルのモデルは、ワクチンや治療薬の開発のためだけでなく、病態の理解に関して、現在HIV - AIDSのための最善の利用可能なモデルです<sup&gt; 1,2,3</sup&gt;。ここで、我々は、細胞性免疫応答の詳細な表現型および機能解析、CD4で特に細胞内サイトカイン産生+およびCD8 + T細胞だけでなく、個々のメモリのサブセットのための方法を説明します。我々は、インターフェロンγ（INF -）とインターロイキン（IL）CD4 +とCD8の両方で-2 PMAプラスイオノマイシン（PMA + I）で刺激したアカゲザルPBMCから+ T細胞の生産のための正確な定量的および定性的な措置を得た。サイトカインプロファイルは、メモリセルの異なるサブセットに異なっていた。さらに、このプロトコルは、我々の研究室で開発されたHIVエンベロープペプチドのカクテルのワクチンで免疫したアカゲザルからPBMCサンプル中に抗原特異的CD4 +およびCD8 + T細胞サブセットの些細な分画を示すために感度を提供する。多色フローサイトメトリー法は、T細胞の異なる集団を正確に識別するための強力なツールです。<sup&gt; 4,5</sup&gt;サイトカイン産生能力を持つ<sup&gt; 6</sup&gt;非特異的または抗原特異的刺激後<sup&gt; 5,7</sup&gt;。

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Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

我々は、合計だけでなく、アカゲザルのCD4 +とCD8 + T細胞、HIV /エイズワクチン研究のための理想的なモデルのメモリのサブセットの詳細な表現型と機能解析のための多色フローサイトメトリーの有用性を実証する。

アカゲザルの細胞性免疫応答のマルチカラーフローサイトメトリー解析

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

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JoVE Journal

Immunology and Infection

21.0K ビュー.  MD Anderson Cancer Center - University of Texas. 我々は、合計だけでなく、アカゲザルのCD4 +とCD8 + T細胞、HIV /エイズワクチン研究のための理想的なモデルのメモリのサブセットの詳細な表現型と機能解析のための多色フローサイトメトリーの有用性を実証する。

ビデオ: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

フローサイトメトリーによる一次免疫細胞亜集団との蛍光ナノ粒子の相互作用を検出

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

サル免疫不全ウイルス特異的CD8の分析<sup&gt; +</sup&gt;ペプチドMHC-I四量体染色によりアカゲザルにおけるT細胞

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

フローサイトメトリーをイメージングを用いた人間システムの免疫シナプスの定性・定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.