この作品は、UNCF -メルク大学院科学研究論文フェローシップ（JLM）、国立心臓、肺、血液研究所の助成金RO1 - HL - 62228（JSJ）とRO1 - HL - 073990（JSJ）によってサポートされていました。

1。ハンクスバランス塩溶液の調製（HBSS） <ol><li>オートクレーブ1Lのガラスビーカー、1000mLのはメスシリンダー、そしてバーをかき混ぜる。 </li><li>再蒸留水で900mLのマークにオートクレーブ1リットルのガラス製ビーカーにHBSS粉のソリューションを再構築。 </li><li>ヘペスのNa +塩3.4 gをとりHBSS溶液を混和し、濃塩酸や水酸化ナトリウムのいずれかを使用して7.35から7.41の間の値にpHを調整する。 </li><li>所望のpHが達成し、安定した状態でされると、アルブミンが完全に溶解するまで室温で溶液にウシ血清アルブミン5gを加える。 </li><li>総量1000 mlのマークになるまでのソリューションに、antimyotic、抗生物質と蒸留水10 mLを加え。 </li><li>徹底的に解決策をかき混ぜる。 </li><li> HBSS溶液をフィルタリングし、後で使用するために50mlのファルコンチューブに分離。追加のチューブが凍結し、後日使用することができます。 </li></ol> 2。動物PreparatioN - 気管チューブの挿入<ol><li>気管チューブ（すなわち、鉗子、はさみ、気管チューブは3-0の黒い編み糸、小動物の人工呼吸器、等）の挿入のために必要な機器や材料でトレイを準備します。 </li><li>ラットは1キシラジン〜2ケタミンの割合でケタミン/キシラジンカクテルと完全に麻酔になると、ラットの腹部、胸部、そして喉を削る。深い麻酔状態が達成されていることを保証するために、ラットのつま先をつまむと四肢のペダルの撤退を確認してください。ラットは、すべての切開を行う前に、深く麻酔であることを確認します。 </li><li> 30 °の角度で傾斜したまな板の上上げ、その頭を持つネズミを配置</li><li>半ば腹部から下顎下に皮膚切開を行います。気管を露出させる前頸三角の筋を離れて解剖。 </li><li>気管の下に3から0黒い編み糸を置きます。 </li><li>軟骨環の間の気管の切開を加えますD気管チューブを挿入します。気管に気管チューブを固定するために緊密に縫合糸を結びます。 </li><li>小動物の人工呼吸器に気管チューブを接続します。 </li></ol> 3。免疫細胞分離のための計器の準備<ol><li>免疫細胞の単離法のための機器（すなわち、鉗子、はさみ、HBSS、針クイックカテーテルを介してソフトテフロン24ゲージバクスター血管内をトリミング）してトレイを準備します。 </li><li> two 10ccの注射器を準備し、心膜嚢と予備のセルを含むHBSSを吸引するためのもう一つのバッファを導入するためのHBSSを使用すると、ワンシリンジを埋める。 3.2.1。分離の手続きのための元の長さの1 / 2〜3 / 4針クイックカテーテル（1.6センチ）以上の24ゲージバクスター血管内のテフロンチップを減らす。 </li><li>すべての注射器、バッファ、および空の15 mLのファルコンチューブ（手続きが終了後、抽出液を保持するため）氷の上に置きます。 </li></ol> &lt;Pクラス=&quot;jove_title&quot;&gt; 4。免疫細胞分離<ol><li>あなたが剣状突起に到達するまで腹壁（白線に沿って）に正中切開を加えます。 </li><li>剣状突起にわずかに横方向の開始、腋窩に向かって斜めに継続的な胸郭を通じて二国間の切開を行います。肺、心臓、または心膜を穿孔しないように注意してください。 </li><li>胸骨の両側の切開が完了したら、内側の側面切削、下部肋骨からダイアフラムを離れて解剖。 注意：心膜が損傷を避けるためにダイアフラムと胸骨に接続されている箇所の今まで注意してください。心膜はダイアフラムの反対側に接続されている場所に注意するランドマークとして、肝鎌状間膜を使用してください。心膜が切断された場合も、細胞を単離する能力は失われます。 </li><li>ゆっくりと胸腔と心を（まだ心膜でカプセル化された）公開する胸郭の片側を持ち上げて。</li><li> HBSSにシリンジを取ると心膜嚢のポイントを見つける胸骨と心臓の間に約半分の方法を（好ましくは吻側尾側より）と心の両方の層を介し短縮クイックカテーテルのテフロンチップを挿入。 </li><li>カテーテルを設置したら、HBBSバッファの2〜3mlで心膜腔を埋める優しく進んでください。あなたが心膜腔が拡大して気づいたときにカテーテルが適切に配置されている場合は、すぐにわかります。ない充填が目撃されていない場合は、クイックカテーテルを撤回し、再配置。 </li><li>心膜嚢がいっぱいになると、他の10 ccシリンジを取ると心に戻って先端を配置し、バッファーを吸引開始。同じエントリの穴を使用することができますまたは別のを作成することができます。特に心臓の下側に、吸引を提供しながら、先端は心膜嚢の壁に近すぎて来ていないことを確認してください。これは、細胞含有細胞の心膜嚢と損失を穿刺する可能性がありますS. </li><li>バッファーを吸引しながら、カテーテルの先端はできるだけ多くのバッファを収集するために、心膜の内部を移動することができます。 </li></ol> 注：もしあなたが少し心を引き裂き、元の穴より大きい心臓の上部に穴を開けて、そうすることで、慌てる必要はありません。限り、心膜はまだ十分な心のを囲むようにあなたはまだバッファより少ない量ではあるが、心臓を経由して別の洗浄を行うことができます。細胞の最適な量のコレクションの場合、3〜4追加の洗浄が必要である。 <ol start="9"><li>氷の上に配置されたファルコンチューブに抽出されたバッファに置きます。 </li><li>さらなる研究のために必要な場合は心臓、肺、および/またはその他の組織を削除します。免疫細胞と他の組織の集まりの後、ラットは、迅速かつ人道的に安楽死でなければならない。 </li><li> 4で200グラム℃で10分間のために収集された免疫細胞のサンプルを遠心常に氷上でチューブを保持しないときは、使用中である。 </li><li> HycloneバッファまたはHBSSを1 mLの上清と再構成ペレットを取り外します。 </li></ol> 注：免疫細胞の単離手順の後、各心臓から得られる免疫細胞の数をカウントすることが重要です。 5。代表的な結果： トルイジンブルーおよびサフラニンO /アルシアンブルーで、この手法で染色を用いて得られた分離された心臓の肥満細胞の例はそれぞれ、図2Aおよび2Bに示されている。図3に、心臓のマストに見られるように、そのような化合物の80分の48とモーガンらに説明されているようにサブスタンスPとして知られている活性剤で処理した後にヒスタミンを放出する能力を維持した。CD4のフローサイトメトリーから9代表的な散布図+細胞はとCD8 +細胞は、それぞれ図4および図5で提示されています。 T -リンパ球は、さらにこの技術から細胞集団として同定と表現型されています。 心臓の免疫細胞の単離手順の図1。フローチャート。 <img alt="図2" src="/files/ftp_upload/3020/3020fig2.jpg" /> トルイジン青（）と（B）サフラニンO /アルシアンブルーである： 図2心臓の肥満細胞の組織学的代表的なイメージは次のように染色されています。。 <img alt="図3" src="/files/ftp_upload/3020/3020fig3.jpg" /> 図3。心臓の肥満細胞の機能研究心外膜心筋部位から分離し、化合物80分の48（10μg/mL）およびサブスタンスP（10μM）で刺激した。 <img alt="図4" src="/files/ftp_upload/3020/3020fig4.jpg" /> 図4。分離されたCD4 +細胞のサブスタンスP（1:100 dilutで標識の代表的なフローサイトメトリーの散布図イオン）。 <img alt="図5" src="/files/ftp_upload/3020/3020fig5.jpg" /> 図5。サブスタンスP（1:100希釈）で標識単離したCD8 +細胞の代表的なフローサイトメトリー。

<ol>
	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+mast+cell+degranulation+prevents+left+ventricular+remodeling+induced+by+chronic+volume+overload+in+rats.">Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.</a> Journal of Cardiac Failure. 11, 548-556 (2005).</li><li>Chancey, A. L., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mast+cell-mediated+activation+of+gelatinase+and+alteration+of+ventricular+diastolic+function.">Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function.</a> Am. J. Physiol. Heart. Circ. Physiol. 282, H2152-H2158 (2002).</li><li>Levick, S. P., Gardner, J. D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, 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=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, 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+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

我々は、開示することは何もない。

心臓の免疫細胞を単離するこのメソッドは通常、伝統的な酵素消化の単離技術の間に発生する不要な活性化することなく、機能、表現型、および成熟度の分析を可能にします。この手法で得られた細胞の平均合計数は、ラットの心臓あたり約40万であり、そしてこれらの細胞の差の染色とフローサイトメトリー分析は、T -リンパ球の混合集団（〜70％）、マクロファージ（〜12％）と肥満細胞?...

<table border="1"><tbody><tr><td>試薬と機器の名称</td><td>会社</td><td>カタログ番号</td><td>コメント</td></tr><tr><td>ハンクス平衡塩</td><td>シグマ</td><td> H4891 </td><td> 1本のボトルにはハンクスバランス塩の1 Lになります ソリューション。再構成ハンクスバランス塩 手順の一部としてソリューション。 </td></tr><tr><td>抗生物質Antimyotic </td><td> GIBCO </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td>シグマ</td><td> H3784 </td><td></td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A3912 </td><td></td></tr><tr><td> 250mLのNalogeneフィルターボトル</td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td>フィッシャーサイエンティフィック</td><td> CAS 7647-01-0 </td><td> 6 Nの場合は、脱イオンにHCLの同じボリュームを追加水。 </td></tr><tr><td> 10NのNaOH </td><td>フィッシャーサイエンティフィック</td><td> SC267 </td><td> 5 Nの場合は、脱イオン水に水酸化ナトリウムの等量を追加。 </td></tr><tr><td>再蒸留水</td><td></td><td></td><td></td></tr></tbody></table>特定の機器の表： <table border="1"><tbody><tr><td>試薬と機器の名称</td><td>会社</td><td>カタログ番号</td><td>コメント</td></tr><tr><td> 3 - O縫合シルク編組</td><td>エチコン製品</td><td> SA64H </td><td></td></tr><tr><td>小動物の人工呼吸器</td><td> CWE株式会社</td><td> SAR - 830 </td><td>分あたり40.5呼吸する呼吸数を設定します。 </td></tr><tr><td>ベベル18ゲージの針</td><td> BDバイオサイエンス</td><td> 305199 </td><td>気管チューブとして使用するための元の長さの1 / 2〜3 ​​/ 4針をトリミング。 </td></tr><tr><td> 24ゲージバクスタークイックカテーテルカテーテルのテフロンチップ（針を除去したもの） </td><td>バクスター</td><td> 2N111 </td><td> 3 / 4〜1 / 2テフロンチップの長さを減らします。 </td></tr><tr><td> 10ccのシリンジ</td><td>フィッシャーサイエンティフィック</td><td> 14から823 - 16B </td><td></td></tr><tr><td> 15 mLのファルコンチューブ</td><td> BDバイオサイエンス</td><td> 352097 </td><td></td></tr><tr><td>ソーバルRT6000B冷却遠心機</td><td>フィッシャーサイエンティフィック</td><td> 75-004-377 </td><td>プレクールに覚えていると4で保つ℃、 </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td>血球計</td><td> Hausserサイエンティフィック</td><td> HS - LB - 1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

。心臓の免疫細胞は肥満細胞、T細胞およびマクロファージを含む1月5日 、これらの免疫細胞は、彼らは多くの心臓疾患の病理学的リモデリングにおいて果たす役割に関心を集めている、店舗やサイトカインを含む生物学的活性のメディエーターの様々を解放し、このようなトリプターゼとしてプロテアーゼ。6-8これらのメディエーターは、マトリックスメタロプロテアーゼの活性化や変調心臓線維芽細胞&quot;機能により、コラーゲンの蓄積を引き起こすことにより細胞外マトリックスの代謝に重要なプレーヤーであることが示されている。9-11しかし、心臓の免疫細胞を単離するために利用できる技術は持っている彼らは心筋組織を消化する細菌性コラゲナーゼを使用するため、問題となって。この手法は、免疫細胞の活性化とそのための機能が失われます。例えば、心臓の肥満細胞が脱顆粒を引き起こす化合物を大幅に軽減応答になる。12だから、我々はtを可能にする技術を開発彼の心疾患におけるこれらの細胞の役割の理解につながる機能性心臓免疫細胞の分離。13、14 このメソッドは、解剖学的なラットの剣状突起、腋窩および鎌状靱帯の位置、および心臓の心膜に精通している必要があります。これらのランドマークは、プロシージャの成功を増加させると心臓の免疫細胞のより高い利回りを確保することが重要です。これらの孤立した心臓の免疫細胞は、機能性、表現型、成熟度、および他の心臓の細胞との共培養実験その相互作用の理解を得るための特性評価に使用することができます。

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

心臓から機能的な免疫細胞を単離するためのこの方法では、これらの細胞の反応性の低下、その結果、不要な免疫細胞の活性化を引き起こすコラゲナーゼ消化、の従来の方法に代わる方法が用意されています。分離の手法は、酵素消化に関連する問題を回避することによって機能的な心臓の免疫細胞が得られます。

機能心臓の免疫細胞の単離

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K ビュー.  University of South Carolina- School of Medicine. 心臓から機能的な免疫細胞を単離するためのこの方法では、これらの細胞の反応性の低下、その結果、不要な免疫細胞の活性化を引き起こすコラゲナーゼ消化、の従来の方法に代わる方法が用意されています。分離の手法は、酵素消化に関連する問題を回避することによって機能的な心臓の免疫細胞が得られます。

ビデオ: Isolation of Functional Cardiac Immune Cells

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

骨髄移植によるマウス大腸炎における免疫および非免疫細胞からの遺伝子発現の機能的役割を差別化

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜由来マスト細胞とその機能特性と Ca2 +の単離-イメージングと脱顆粒の試金

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...