この作品は、カナダの協会の健康調査 (148808 148792) によって支えられた、SE に支えられ心臓・卒中財団、オンタリオ地方事務所、10 セントの行進財団、テッド ・ ロジャース センターの心研究 Peter Munk 人材賞」を受賞心臓センター。XCC 機構バンティング交わりを保持します。ラは、心臓と脳卒中/リチャード ・ Lewar 就労賞を保持します。

倫理ステートメント: このプロトコルがレビューおよび大学健康ネットワーク (トロント、カナダ) で動物ケア委員会によって承認された動物のケアに関するカナダの理事会に準拠して。
1. バッファー準備
<ol><li>HBB バッファー (ハンクのバランスの取れた塩ソリューション (HBSS)、2% の熱不活化ウシ血清、0.2% ウシ血清アルブミン) を準備します。10 mL の熱不活ウシ血清と HBSS の 500 mL にウシ血清アルブミン 1 g を追加します。フィルターは 0.2 μ m のフィルターを通して消毒し、4 ° C で保存</li>
	<li>FACS バッファーを準備 (リン酸緩衝生理食塩水 (PBS)、2% の熱不活化牛血清、エチレンジアミン四酢酸 (EDTA、pH 8.0) 1 mM)。10 mL の熱不活ウシ血清と 500 mL の PBS に 0.5 M の EDTA の 1 mL を追加します。フィルターは 0.2 μ m のフィルターを通して消毒し、4 ° C で保存 注: ソリューションは 4 ° C で 1 ヶ月保存できます。</li>
</ol>2. 白血球の循環のラベリング
<ol><li>無菌 1 μ g 滅菌リン酸緩衝生理食塩水 (PBS) 200 μ L/マウスの最終的な注入量で希釈した抗体の抗マウス CD45 注入を準備します。28.5 G インスリン注射器にボリュームをロードし、光から離れた保ちます。 注: は、(消化後染色; ステップ 4 を参照してください)、心内染色から血管 (静脈投与による染色) を区別するために 2 つの異なる蛍光標識抗 CD45 抗体を使用します。</li>
	<li>
あらかじめ 5 分赤熱ランプに尻尾を公開することによってマウス (8-12 週齢) を温めます。 注意: は、動物を過熱しません。ランプを安全な距離を保つ (> 30 cm)。<ol>
<li>適切なデバイスを使用して胸骨の位置で動物を抑制します。公開し、人差し指と中指で尾の根元、親指と薬指で尾の半ば遠位領域を固定化した非支配的な手と尻尾を安定させます。 注意: は、皮膚引っ張ることができると尾の先端であまりにも多くの力を適用されません。ベベルの静脈に上向きの針を挿入します。静脈を穿刺しやすいように針を常時静脈と平行にしてください。 注記: 注射器に流れ込む血液が発生し、良い注入の印であります。針を 90 ° の角度に曲げ投与を容易にすることができます。曲げ針が危険を表すことができるよう、安全管理室に確認します。</li>
		<li>200 μ L を尾静脈静脈内注入します。流体は、容易に流れるし、静脈を一時的に解除ください。 注意: 抵抗を感じる場合は、液を押し込まないでください。これは、針の紛失のサインです。</li>
		<li>マウスを人道的に安楽死させる前に 5 分間循環する抗体を許可します。</li>
	</ol>
</li>
</ol>3. 心を分離して消化
<ol><li>
CO2を用いたマウスを安楽死させる、または他の安楽死制度受け入れられたプロシージャ。
	<ol>
<li>CO2安楽死、CO2チャンバーにマウスを置き、毎分 (すなわち、商工会議所は 10 L、2 L/分で埋める場合) 室容積の 20% にフィル レートを設定します。呼吸が停止しているまで 2 分とモニター マウスを実行します。</li>
		<li>CO2オフにシャット ダウンし、追加の 1-2 分商工会議所を開く前に呼吸が停止していることを確認マウスを残してつま先ピンチ マウスがマウスの処理に進む前に死んだことを確認するを実行します。</li>
	</ol>
</li>
	<li>70% のエタノールとマウスをスプレーします。腹部の皮膚を持ち上げて、胸骨まで腰のレベルで腹側正中初めに沿って切断することによってマウスを解剖します。腹部を開き横隔膜を公開する肝臓を移動します。</li>
	<li>
肺と心臓を刺さないように注意しながら、正中線の両側に、横隔膜、肋骨をカットします。
	<ol>
<li>剥離による心臓当時は肋骨、左心房、右心房続いてカット最初を公開します。 注意: は、この臓器に含まれている血液のフラッシュの効率が低下することができます、動脈や静脈、負傷者なしを確保、心房をカットするときに注意を使用します。</li>
		<li>15-20 ml 21 G 針装備 60 mL の注射器を使用して冷たい PBS と冷 PBS の心臓を灌流します。優しく鉗子で心の底をつかみ、左の心室の頂点近くに針を挿入します。右心室の手順を繰り返します。適切な注入は、白い肺と淡い肝臓の結果します。 注: 灌流は、血液凝固は、細胞を循環と利子の器官を汚染することができますを避けるために安楽死した後 5 分以内で実行する必要があります。</li>
	</ol>
</li>
	<li>
鉗子で心のベースを保持し、優しく心を引き上げて、主要な肺動脈と静脈、大動脈、心膜の袋を切断することによって心をデタッチします。
	<ol>
<li>きれいな糸くずペーパー タオルで優しく親指と人差し指の間心を保持することによって、心をきれいにし鉗子と心房と主要な動脈と静脈および他の汚染物質の残党をやってのけます。 注: は、心房全体が削除され、肺や血管などの他の組織が存在しないこれらの組織が自分の居住者の免疫細胞を持っているを確認します。肺特に心筋内のそれらのより小さい人口をマスクすることができます大規模な免疫細胞の人口があります。</li>
	</ol>
</li>
	<li>
冷 PBS の 50 μ L で 5 cm のプラスチックの皿の中心を配置します。
	<ol>
<li>表示部分がない、滑らかな整合性まで湾曲した解剖はさみを使用して心臓をミンチします。 注: は、組織の損失を最小化するまな板中皿の含まれる領域で心を維持してください。</li>
		<li>2 mL 遠心チューブ 800 μ L 冷たいダルベッコ変更イーグル培地 (DMEM) とにカーブタイプ鉗子を用いた thetrituratedheart 組織に転送します。</li>
	</ol>
</li>
	<li>
私、ヒアルロニダーゼ型私 - s、60 U/mL と DNase の 60 U/mL 450 U/mL ofcollagenase を追加-各サンプルに。
	<ol>
<li>45-60 分間で 50 rpm ロッキング シェーカーで 37 ° C でサンプルをインキュベートします。</li>
	</ol>
</li>
	<li>
消化力の渦のサンプルに簡潔に後 (20 s)、すぐに 4 ° C でサンプルを保つために氷の上
	<ol>
<li>組織サンプルが均一、ピペット チップを詰まらせるれませんまで上下サンプルのピペット 1 mL pipettor を使用します。 注: は、再現性を高める可能性がありますピペットに特定回数を定義します。心は最適な消化されない場合心筋の部分ピペットを妨害する可能性があります。遠心チューブに対してピペット先端を軽く研削と、組織の大きな部分を均質化に役立ちます。</li>
	</ol>
</li>
	<li>
あらかじめ冷たい HBB の 1 mL を 50 mL の円錐管に 40 μ m セル ストレーナーを濡れています。
	<ol>
<li>フィルターに均質化サンプルを追加し、ゆっくり冷たい HBB の 12-14 mL を注いでを介して洗浄します。</li>
		<li>4 ° C でラベルの 15 mL の円錐管にフィルターが適用されたサンプルを転送し、</li>
	</ol>
</li>
	<li>
4 ° C で 5 分間 400 × gで遠心分離機サンプル上清を離れて吸い出しなさい。
	<ol>
<li>各サンプルに 1 mL のアンモニウム塩化カリウム (ACK) 換散バッファーを追加することで赤血球を溶解させます。ボルテックスによってサンプルを再懸濁します、5 分間室温でインキュベートします。</li>
		<li>溶解が完了すると、5-8 mL の冷たいハンクのバランスの取れた塩ソリューション (HBSS) 溶血を停止するを追加します。</li>
	</ol>
</li>
	<li>遠心分離機の 400 x「アウトドア 4 ° c で 5 分間のサンプル上清を離れて吸い出しなさい。</li>
	<li>各サンプルとラベル付き 1.5 mL 遠心チューブへの転送に 1 mL の FACS バッファーを追加します。 注: プロトコルを一時停止できるここで 2 h までの 4 ° C で保存のサンプル</li>
</ol>4 流れの Cytometry の染色と分析
<ol><li>
4 ° C で 5 分間 400 × gで遠心分離機サンプル
	<ol>
<li>非特異抗体の結合を減らすためには、1: 100 抗 CD16/CD32 および単球ブロック (1:20) を希釈サンプルあたり 50 μ L FACS 会計 (材料の表を参照)。15 分間室温でインキュベートします。</li>
		<li>50 μ L のサンプル/FACS のため抗体希釈 (各抗体 1: 250) 会計を準備します。 注: 生存率色素抗体カクテルで死んだ細胞を除外するために含めることができます。また、使用される (図 1CとD)、流れ解析による小型除外もあります。</li>
		<li>洗浄せず抗体各サンプルし、ミックス マスター ミックスの 50 μ L を追加します。</li>
		<li>暗闇の中で 30 分間、4 ° C でサンプルをインキュベートします。</li>
	</ol>
</li>
	<li>洗浄し、遠心分離機の 4 ° C で 5 分間 400 x gでサンプルごとのサンプルを 600 μ L FACS を追加します。</li>
	<li>上清を吸引し、300 μ L FACS でペレットを再懸濁します。暗闇の中で 4 ° c のサンプルを保ちます。 注: フローサイトメトリーを実行する前に 10 分以内で染色 DAPI など、いくつかの生存の染料が必要です。</li>
	<li>フローサイトメトリーによる分析 (図 1 および図 2を参照) 次の 3 時間以内。長い時間が必要な短期的固定低濃度パラホルムアルデヒドを使用して場合 (0.5-1 %pbs) 染色を維持するためにお勧めします。 注: 開始フロー フローサイトメトリー分析の前に 40 μ m セル ストレーナー流れの cytometer の流体工学の妨害を防ぐためにサンプルのフィルターを適用することを勧めします。</li>
</ol>

<ol>
	<li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+natural+history+of+human+myocarditis.">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li><li>Epelman, S., 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=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Pinto, A. R., 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=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814 (2012).</li><li>Lavine, K. J., 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=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+function+of+cardiac+macrophages.">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li><li>Hulsmans, M., 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=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Nahrendorf, M., 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=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li><li>Clemente-Casares, X., 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=A+CD103(+)+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis.">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li><li>Van der Borght, K., 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=Myocardial+Infarction+Primes+Autoreactive+T+Cells+through+Activation+of+Dendritic+Cells.">Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.</a> Cell Reports. 18 (12), 3005-3017 (2017).</li><li>Galkina, E., 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=Preferential+migration+of+effector+CD8++T+cells+into+the+interstitium+of+the+normal+lung.">Preferential migration of effector CD8+ T cells into the interstitium of the normal lung.</a> Journal of Clinical Investigation. 115 (12), 3473-3483 (2005).</li><li>Edelson, B. 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=Peripheral+CD103++dendritic+cells+form+a+unified+subset+developmentally+related+to+CD8alpha++conventional+dendritic+cells.">Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells.</a> Journal of Experimental Medicine. 207 (4), 823-836 (2010).</li></ol>

著者が明らかに何もありません。

心筋の炎症や心筋炎、最も心血管疾患の機能であります。ただし、心筋はありません非病気の状態で、独自の免疫のコンポーネントを欠いています。定常状態の時に多くの免疫細胞は、心筋に存在し、保守と保護の不可欠な役割を果たします。細胞のこれらの多様な人口の特性はこのプロトコルで発表したものなどの方法なしで可能をでしょう。
心筋の機械的および酵?...

心筋応力または損傷、虚血を含む (虚血再灌流や心筋梗塞) の異なった形態と非虚血性 (高血圧や心筋炎) も修復と保護、炎症細胞の募集を促進します。病原性のプロパティ。1891 年には早くもラコストは最初に発疹チフス、猩紅熱の1に感染した患者の心筋細胞の浸潤の存在を説明しました。ただし、心筋免疫細胞の詳細な研究に必要なより高度な診療技術の開発。その結果、ごく最近我々 は定常状態の間に本質的なメンテナンスの役割を持つ免疫細胞の多様な人口が心筋内に存在することを理解し始めています。
組織されているとまだ、炎症における免疫細胞を特徴付ける最も一般的な方法です。しかし、組織は、貴重な診断ツール、心の免疫細胞の研究での使用で重要な制限があります。心筋に存在する免疫細胞は総細胞の非常に小さい割合を表すし、比例して巨大空間でもっとまたはより少なく均等に分散します。同様に、ウイルス性心筋炎など、心筋の炎症のいくつかのフォームは、炎症の焦点のパターンを示します。つまりは心の免疫システムの正確な分析がしばしば組織学的サンプリング、バイアスを減らすためのコストの増加の高い学位を必要とします。また、組織は、細胞の識別 (例えばパラメーターの数) で使用可能な情報の非常に限られた量を提供します。細胞サブセット (表面マーカー、転写因子分泌分子など) 複数の式マーカーの使用を必要とする数が増え、フローサイトメトリーは、診療の最も強力なツールとして地位を確立して低コスト、高スループット。
診療技術の使用は多数細胞の単一細胞分析のできる効率的な消化力のプロトコルの開発に密接に依存します。細胞抽出の徹底的なプロトコルの開発は、心臓の免疫学の研究で機会の新しいウィンドウを開きます。消化、フローサイトメトリー、トランスクリプトームの組み合わせを介してマクロファージと樹状細胞の中心部に存在する主要な人口は特徴付けられる2,3をされています。安定状態で心臓の免疫細胞の最も豊富な人口が CD64+MerTK+ CCR2 または CD11c2の表現に基づいてさらに分けることができますマクロファージ。CCR2+に対し、マクロファージが成人の骨髄造血に属します大半 CCR2 の MHC-II の高または低のレベルを表現できる、-マクロファージが出生前の起源の主に。マクロファージは、修理4傷害または定常状態における電気的接続6のサポート中に破片5の除去など主要な機能を実行します。異なるフォームの炎症の時に Ly6CこんにちはMHC-IIlo CD64int単球の重要な流入発生、これは後に Ly6CこんにちはCCR2 分化+マクロファージ2,7.健常者の心筋に存在する細胞のかなり小さいが重要な人口樹状細胞8,9で構成されます。心臓従来 Dc (cDCs) の 2 つの主要なサブセットが最近特徴付けられる: cDC1 (CD103+ Dc)、cDC2 (CD11b+ Dc)。Dc は感染8に対して防衛で重要な役割を果たすが、促進炎症、心筋梗塞9特に中に自傷をすることも。
ここでは、単純なマウス心筋から心臓免疫細胞の分離法について述べる.メソッドは、分析、または流れの cytometry の並べ替えまたは磁気ビーズ濃縮により更に精製できる単一細胞懸濁液を得るためにセル ストレーナー濾過と酵素的及び機械的消化を組み合わせたものです。管外の心筋細胞の正確な測定は、心臓の血管の血流からの可能な汚染物を除去するために心筋灌流を必要があります。さらに、さらに撮影らプロトコル10に基づく血管内の汚染物質から心筋細胞を区別する使用ことができます免疫細胞の血管内ラベリングのオプションの手順を提案する.最後に、主要なマクロファージと cDC のサブポピュレーションを識別するために基本的な流れフローサイトメトリー解析を提案します。

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse: Batf3-/-</td>
			<td>The Jackson Laboratory</td>
			<td>JAX: 013755</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of extravascular immune cells of the heart

免疫システムは、健康な心臓の重要なコンポーネントです。心筋は、定常時および炎症の異なった形態の間に機能区分と異なる免疫細胞サブセットの豊富な人口に家。最近まで、心で免疫細胞の研究に必要な顕微鏡を使用または小さな未発達の消化力のプロトコル、重度の炎症の中に十分な感度を提供が、自信を持ってすることができませんでしたを識別する-キーですが-の人口安定状態でセルです。ここでは、簡便な合成酵素 (コラゲナーゼ、ヒアルロニダーゼや DNAse) と前に小を区別する蛍光標識抗体の血管内投与マウスの心の機械的消化について述べるが、やむを得ない血管内細胞の汚染物質。このメソッドは、識別、表現型解析と定量化、フローサイトメトリーで分析したまたは蛍光活性化細胞ソーティングまたは磁気ビードの分離のために更に精製できる分離の実行可能な細胞の懸濁液を生成します転写の解析または生体外で研究。キーのマクロファージと樹状細胞集団の中心部を区別するためにステップバイ ステップ フローサイトの例が含まれています。中規模実験 (10 心) プロシージャの完了には 2-3 h が必要です。

日には、よい方法設計されていますない心筋細胞など、他のコンポーネントの心筋細胞から免疫細胞を分離します。したがって、CD45 続く単一セルおよび小型除外ゲート (図 1A) 免疫細胞群を識別するために、あらかじめゲーティング フローサイトメトリーによる心筋単一細胞懸濁液の分析が必要です。また、死んだ細胞を除外する?...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

心の血管外免疫細胞の分離と同定

このプロトコルを分離、識別し、定常状態や炎症の間にマウスの心筋に存在する免疫細胞を定量化するシンプルかつ効率的な手法を提案します。プロトコルは、フローサイトメトリーによるさらに分析することができます単一細胞懸濁液の生成のための酵素的及び機械的消化を組み合わせたものです。

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Research

JoVE Journal

Immunology and Infection

12.4K ビュー.  University Health Network (UHN). このプロトコルを分離、識別し、定常状態や炎症の間にマウスの心筋に存在する免疫細胞を定量化するシンプルかつ効率的な手法を提案します。プロトコルは、フローサイトメトリーによるさらに分析することができます単一細胞懸濁液の生成のための酵素的及び機械的消化を組み合わせたものです。

ビデオ: Isolation and Identification of Extravascular Immune Cells of the Heart

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 ...

Isolation of Functional Cardiac Immune Cells

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 include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

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 ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...