Name: isolation and in vitro culture of murine and human alveolar macrophages
Uploaded: 2018-04-20T14:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

原稿を編集の支援ありがとうクレア プレンダーガスト。DKN はサポートされて研究でフリン財団から (#2095) を付与し、TM は国立衛生研究所 (R01HL056643 と R01HL092514) からの補助金によってサポートされています。DKN 手法を開発、研究をデザイン、そして書いた原稿;動物実験と臨床サンプル調達支援 OMSB 支援フローサイトメトリー解析・細胞のソーティング。TM は研究を監督し、原稿の見直し。

ここで説明したすべてのメソッドは、機関動物ケアおよび使用委員会 (IACUC) セントジョセフ病院と医療センター制度レビュー委員会 (IRB) によって承認されています。
1. マウスの気管支肺胞洗浄 (BAL) 液から AMs の分離
<ol><li>ケタミン (87.5 mg/kg 体重) とキシラジン (12.5 mg/kg 体重) 腹腔内注射を介してカクテル 8 週齢 c57bl/6 マウスを anaesthetize します。マウスに達する反射の損失や筋肉の弛緩で外科麻酔を進みます。</li>
	<li>郭清の面に腹側を上にマウスを配置します。脱水症状を防ぐために、滅菌アルコール準備を 70% イソプロパノール消毒すると飽和するパッドとマウスの腹側表面全体を拭く目に獣医眼科用軟膏を適用します。</li>
	<li>拘束された 4 本の脚を持つマウスをマウントします。</li>
	<li>優しく顕微解剖ツールで腹腔内を開きます。すべての内臓の損傷や張り出した組織を作成しなくてもできるだけ同様に多くの領域を開くには注意が必要があります。</li>
	<li>優しくゆっくりと縦隔から胸郭を切開して胸腔内を開きます。理想的には、肋骨は側に側から摘出することができます。胸腔内を開くときの肺の胸膜を傷つけないように十分な注意が必要があります。</li>
	<li>下大静脈を探し、マウスを出血する切開をおきます。血液を吸収する吸収性綿パッドを使用します。</li>
	<li>循環血液細胞をフラッシュする (25 G 針、10 mL の注射器を使用して) 右心室に 10 mL の冷たいリン酸緩衝生理食塩水 (PBS) を挿入します。吸収性綿パッドとは、血液の流れを吸収します。完全に灌流、肺湯通し表示され、洗浄の準備ができているし。</li>
	<li>優しく皮膚を開いてカットし、気道を公開する首の筋肉します。慎重に気管を損傷することがなく隣接する筋肉、軟骨、脂肪組織を切除します。</li>
	<li>小さな切開 (< 2 mm) 気管喉頭の後方に。この切開は気管、喉頭にまだ接続されている間にカテーテルを挿入するだけで十分なはずです。針なし 1 インチ 22 G カテーテルを肺に向かって気管に挿入します。シルク編み縫合糸でカテーテルを固定 (4-0; 非吸収性) 正方形の結び目を持つ。 注: カテーテルが整う必要がマウスのサイズに基づいて。 ある非常に短期または長期のカテーテルは、リークおよび/または洗浄中に気道を引き裂く傾向があります。カテーテルの長さはこのようなする必要があるときに完全に挿入される先端まま気管内も、主気管支に到達します。</li>
	<li>冷たいバル バッファー (Ca2 +と Mg2 +無料 PBS + 1 mM EDTA エチレンジアミン四酢酸) カテーテルでいっぱい 1 mL 注射器を接続し、ゆっくりと肺の中にバッファーを植え付けます。これは肺を膨らませます。5 秒間、カテーテルに接続されている注射器を維持し、軽くピストンを引いて、洗浄液を吸引します。これは肺に空気を抜きます。氷上に置いた 15 mL の円錐管に流体を収集します。</li>
	<li>さらに 9 回のステップは 1.10 を繰り返し、洗浄液をプールします。肺の容量を超えるし、その破裂につながる可能性がありますので、フラッシュあたり 1 mL のバッファーを超えないようにします。</li>
	<li>IACUC 承認プロトコルによって CO2の過剰摂取とマウスを安楽死させます。</li>
	<li>10 分の 4 ° C で 250 x g で 10 mL BAL 液を遠心分離機します。細胞ペレットには、バルのセルが含まれています。 注意:ペレットは非常に小さく、上清を吸引するときので注意します。</li>
	<li>流れ/並べ替えバッファー (PBS + 2% 不活化熱牛胎児血清 (FBS) + 1 mM EDTA 25 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulphonic 酸 (HEPES)) の 100 μ L の BAL 細胞を再懸濁します。</li>
	<li>マウス Fc ブロックの追加により、Fc 受容体に非特異的な抗体の結合をブロック (2.4G2、10 μ g/mL のクローン) 氷の上 20 分間インキュベートし、。</li>
	<li>抗体と一緒にマイナス 1 (FMO) 蛍光染色を実行し、単一染色コントロール11,25。BAL を評価セル アナライザーによって細胞が適切な分析ソフトウェア単一セル フローサイトメトリー分析による続く (材料の表を参照してください) (材料の表を参照してください)。</li>
	<li>AMs を分離蛍光活性化セルによる選別機 (材料の表を参照してください) 直接 1 mL マウスに時に培地の培養または生体内でAMs は、携帯転送。また、1 mL の溶解バッファーに並べ替え AMs は 10 μ L/mL β-メルカプトエタノール RNA の抽出で補完 (材料の表を参照してください)。</li>
</ol>2. AMs マウス肺、単一細胞懸濁液からの分離
<ol><li>ケタミン (87.5 mg/kg 体重) とキシラジン (12.5 mg/kg 体重) 腹腔内注射を介してカクテル 8 週齢 c57bl/6 マウスを anaesthetize します。マウスに達する反射の損失や筋肉の弛緩で外科麻酔を進みます。</li>
	<li>郭清の面に腹側を上にマウスを配置します。脱水症状を防ぐために、70% イソプロパノール消毒すると飽和滅菌アルコール パッドとマウスの腹側表面全体を拭く目に獣医眼科用軟膏を適用します。</li>
	<li>拘束された 4 本の脚を持つマウスをマウントします。</li>
	<li>優しく顕微解剖ツールで腹腔内を開きます。すべての内臓の損傷や張り出した組織を作成しなくてもできるだけ同様に多くの領域を開くには注意が必要があります。</li>
	<li>優しくゆっくりと縦隔から胸郭を切開して胸腔内を開きます。理想的には、肋骨は側に側から摘出することができます。胸腔内を開くときの肺の胸膜を傷つけないように十分な注意が必要があります。</li>
	<li>下大静脈を探し、マウスを出血する切開をおきます。血液を吸収する吸収性綿パッドを使用します。</li>
	<li>循環血液細胞をフラッシュする (25 G 針、10 mL の注射器を使用して) 右心室に 10 mL の氷冷 PBS を挿入します。完全に灌流、肺湯通し表示され、収穫の準備ができています。</li>
	<li>風邪の 5 mL と 15 mL の円錐管に気管、血管、靭帯を切断して肺を解剖ダルベッコ変更イーグル培地 (DMEM)。次のステップまで氷の上それを維持します。</li>
	<li>IACUC 承認プロトコルによって CO2の過剰摂取とマウスを安楽死させます。</li>
	<li>3 ml DMEM 無菌、パイロジェン フリーの 60 mm ディッシュに灌流の肺を転送します。鉗子を解剖の助けを借りて、航空および他のハードの非肺組織材料をいじめる存在する場合。肺組織にメスをミンチ < 1 mm サイズ。</li>
	<li>300 μ g/mL Liberase TL と 5 U/mL 追加 DNase I、ピペッティングで混ぜるし、インキュベーターの 37 ° C で 25 分間インキュベートします。軽く 10 分後ピペッティングにより一度混ぜます。</li>
	<li>50 mL コニカル チューブにインストールされている 100 μ m セル ストレーナーを解離肺を通過します。1 mL 注射器からプランジャーの後ろを使用すると、フィルターの細胞塊をマッシュ アップします。20 mL 洗浄バッファーが付いているフィルターを洗う (PBS + 2% 熱不活化の FBS + 2 ミリメートルの EDTA)。</li>
	<li>4 ° C で 500 × g で 5 分間遠心します。上澄みを廃棄し、20 mL 洗浄バッファーで細胞ペレットを再懸濁します。</li>
	<li>手順 2.12 と二度、2.13 たびにフィルターとチューブを変更するを繰り返します。5 分以上の洗浄バッファーのフィルターを予浸午前収量が増加します。単一細胞懸濁液を準備するには、追加し 100 μ L 細胞ペレットにバッファーの流れ/並べ替え。</li>
	<li>マウス Fc ブロックの追加により、Fc 受容体に非特異的な抗体の結合をブロック (2.4G2、10 μ g/mL のクローン) 氷の上 20 分間インキュベートし、。</li>
	<li>FMO と単一染色コントロール11,25染色抗体を実行します。肺を評価セル アナライザーによる細胞単一セル フローサイトメトリーによる解析ソフトウェア後 (材料の表を参照してください)。</li>
	<li>セルの並べ替えで並べ替え AMs (材料の表を参照してください) 直接 1 mL マウスに時に培養培地の培養または生体内でAMs は、携帯転送。また、1 mL の 10 μ L/mL β-メルカプトエタノール RNA 抽出のための補足の換散バッファーに AMs を並べ替えます。</li>
</ol>3. 人間の BAL 液から AMs の分離
<ol><li>4 ° C で研究室に診療所から人間の BAL 液の送迎を手配します。</li>
	<li>BAL 液 (10-50 mL) を 4 ° C で 250 x g で 10 分間遠心します。細胞ペレットには、BAL 細胞11が含まれています。</li>
	<li>20 mL 洗浄バッファーの餌を洗浄 (PBS + 2% 熱不活化の FBS + 2 ミリメートルの EDTA)、4 ° C で 250 x g で 10 分間遠心します。</li>
	<li>フロー/並べ替えバッファーの 100 μ L、BAL 細胞を再懸濁します (PBS + 2% 熱不活化 FBS + 1 mM EDTA 25 mM HEPES)。</li>
	<li>人間の Fc ブロック (クローン Fc1.3070、25 μ g/mL) を追加し、氷で 20 分間インキュベート Fc 受容体に非特異的な抗体の結合をブロックします。</li>
	<li>FMO と単一染色コントロール11,24を汚す抗体を実行します。BAL を評価セル アナライザーによる細胞単一セル フローサイトメトリーによる解析ソフトウェア後 (材料の表を参照してください)。</li>
	<li>セルソーターによる並べ替え AMs (材料の表を参照してください) 直接培地 1 mL 人間午前に 1 mL の溶解バッファーを添加した 10 μ L/mL β-メルカプトエタノール。</li>
</ol>4. 文化体外AMs の
<ol><li>次のセルの並べ替え、4 ° C 10 分で 250 × g で遠心分離によってセルを収穫します。</li>
	<li>1 mL人間午前培[ロズウェル パーク記念研究所中 (RPMI) 10% を添加した 1640 FBS、20 %l-929 文化 (マクロファージコロニー刺激因子 (M-CSF)、100 U/mL 最終濃度ソース) として上清で人間の AMs を再懸濁します1 mM ピルビン酸ナトリウム、N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic 酸 (HEPES) 10 mM、1 x ペニシリン/ストレプトマイシン (省略可能)]。同様に、1 mLマウス午前培[10 %fbs、20 %l-929 培養上清 (100 U/mL 最終濃度 M CSF ソース) として 1 mM ピルビン酸ナトリウム、10 mM HEPES、1 x ペニシリン/ストレプトマイシン (オプションと DMEM で AMs マウスを再懸濁します)].</li>
	<li>細胞カウンターによって生菌トリパン ブルーを除くを列挙し、1 x 106/mL で実行可能な細胞濃度を調整します。 注: 理想的を 5 × 105 AMs から分離でき、8-10 週古い C57BL/6 の雄マウスから収集した BAL 液。マウス肺ダイジェストから午前の収穫は可変してマウス BAL 液のそれよりもやや少ない可能性があります。人間の BAL 液から午前の収穫は、BAL 液、洗浄、および患者の臨床状態で使用されている生理食塩水の総容積の量によって異なります。</li>
	<li>チャンバー スライド、培養皿または培養フラスコ内のセルの種子収量と実験の必要性に基づき。理想的には、T25 培養フラスコに 10 mL の培地で 1 × 105/mL で AMs をシードします。</li>
	<li>セル 5% CO2雰囲気の 37 ° C の加湿インキュベーターで孵化させなさい。 注: AMs 付着性のセルとして成長し、非常にゆっくりと成長 (倍加時間 > 7 日間)。</li>
	<li>播種後 24 時間で AMs すべき付着と培地の穏やかな変化ではデタッチされません。端から端まで吸引してメディアを削除し、37 ° C に加温新鮮な培地で補充します。 注: セルは、午前生理学または刺激の効果を評価するために今すぐ使用できます。AMs チャンバー スライドで成長に適切な抗体便利に汚すことができるし、可視化に最適です。流れフローサイトメトリー評価、AMs はソリューションを解離非酵素的セル孵化によって収穫されています。</li>
	<li>培養液を吸引し、文化面 (T25 フラスコあたり約 2 mL) をカバーし、37 ° C で 5 ~ 10 分間インキュベートする十分な分離液を追加します。削りが不要になると機械的に細胞をこすり取る際に勧めはしませんが。側面を軽く 1-2 mL の流れ/並べ替えバッファーを追加して、優しく上下に付着性のセルの大部分をデタッチするピペットします。</li>
	<li>RNA 研究の換散バッファーを追加して培養皿に直接細胞を溶解します。</li>
</ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+alveolar+proteinosis:+a+bench-to-bedside+story+of+granulocyte-macrophage+colony-stimulating+factor+dysfunction.">Pulmonary alveolar proteinosis: a bench-to-bedside story of granulocyte-macrophage colony-stimulating factor dysfunction.</a> Chest. 136 (2), 571-577 (2009).</li><li>Happle, C., 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=Pulmonary+transplantation+of+macrophage+progenitors+as+effective+and+long-lasting+therapy+for+hereditary+pulmonary+alveolar+proteinosis.">Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.</a> Sci Transl Med. 6 (250), 250ra113 (2014).</li><li>Suzuki, 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=Pulmonary+macrophage+transplantation+therapy.">Pulmonary macrophage transplantation therapy.</a> Nature. 514 (7523), 450-454 (2014).</li><li>Hashimoto, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+self-maintain+locally+throughout+adult+life+with+minimal+contribution+from+circulating+monocytes.">Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.</a> Immunity. 38 (4), 792-804 (2013).</li><li>Murphy, J., Summer, R., Wilson, A. A., Kotton, D. N., Fine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prolonged+life-span+of+alveolar+macrophages.">The prolonged life-span of alveolar macrophages.</a> Am J Respir Cell Mol Biol. 38 (4), 380-385 (2008).</li><li>Maus, U. A., 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=Resident+alveolar+macrophages+are+replaced+by+recruited+monocytes+in+response+to+endotoxin-induced+lung+inflammation.">Resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation.</a> Am J Respir Cell Mol Biol. 35 (2), 227-235 (2006).</li><li>Perdiguero, G. 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=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Bitterman, P. B., Saltzman, L. E., Adelberg, S., Ferrans, V. J., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophage+replication.+One+mechanism+for+the+expansion+of+the+mononuclear+phagocyte+population+in+the+chronically+inflamed+lung.">Alveolar macrophage replication. One mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung.</a> J Clin Invest. 74 (2), 460-469 (1984).</li><li>Misharin, A. V., 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=Monocyte-derived+alveolar+macrophages+drive+lung+fibrosis+and+persist+in+the+lung+over+the+life+span.">Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span.</a> J Exp Med. , (2017).</li><li>Zheng, Z., 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=Donor+pulmonary+intravascular+nonclassical+monocytes+recruit+recipient+neutrophils+and+mediate+primary+lung+allograft+dysfunction.">Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction.</a> Sci Transl Med. 9 (394), (2017).</li><li>Nayak, D. 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=Zbtb7a+induction+in+alveolar+macrophages+is+implicated+in+anti-HLA-mediated+lung+allograft+rejection.">Zbtb7a induction in alveolar macrophages is implicated in anti-HLA-mediated lung allograft rejection.</a> Sci Transl Med. 9 (398), (2017).</li><li>Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R., Perlman, H. <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+macrophages+and+dendritic+cell+subsets+in+the+mouse+lung.">Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung.</a> Am J Respir Cell Mol Biol. 49 (4), 503-510 (2013).</li><li>Kopf, M., Schneider, C., Nobs, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+function+of+lung-resident+macrophages+and+dendritic+cells.">The development and function of lung-resident macrophages and dendritic cells.</a> Nat Immunol. 16 (1), 36-44 (2015).</li><li>Eguiluz-Gracia, I., 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=Long-term+persistence+of+human+donor+alveolar+macrophages+in+lung+transplant+recipients.">Long-term persistence of human donor alveolar macrophages in lung transplant recipients.</a> Thorax. 71 (11), 1006-1011 (2016).</li><li>Yu, Y. A., 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=Flow+cytometric+analysis+of+myeloid+cells+in+human+blood,+bronchoalveolar+lavage,+and+lung+tissues.">Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues.</a> Am J Respir Cell Mol Biol. , (2015).</li></ol>

著者の利害の衝突を宣言するあります。

AMs は、肺に生じる出生時と26全体の寿命を永続的な作成時間の長いリビング肺居住者マクロファージです。肺生理学7と病理12とその潜在的な肺の免疫24の予測における役割を認識しています。AMs 肺11,27で長期的な存在感があるので、活性化と免疫11,<sup...

肺微小環境は精巧な空気の導管と血管を一意に複雑な生態系です。吸入空気は、血液-空気ガス交換が発生する、肺胞に達する前に気管と気管支、細気管支の多数の枝を通してを移動します。大気と直接相互作用は、呼吸の表面は浮遊粒子状物質や汚染物質の可能性のある有害な効果から保護を必要とします。物理的、化学的、免疫学的障壁の数は、肺を保護します。特に、食細胞呼吸面での展開は、重要な最初の行の防衛システムを提供しています。肺胞マクロファージ (AMs) 肺居住者食細胞の一種であるし、彼らは肺マクロファージ プールの大半を構成します。その名の通り、AMs は主に肺胞腔に局在しているし、常に周囲の雰囲気をサンプルし、歯槽の上皮1通信付着細胞として発生します。肺胞腔内の食細胞の 95% 以上は、定常状態の肺、AMs2、その組成は、炎症、感染症、または汚染物質への慢性露出により変わる可能性があります。
AMs は、全身の重要性のおよび/または肺に対してローカルにすることができる機能の広い範囲に参加します。例えば、AMs は肺の最適な機能と開発に不可欠です。免疫の監視;侵入する病原体と吸入粒子3,4,5,6,7細胞の残骸のクリアランス。AMs の対象の枯渇は、呼吸器系ウイルスや細菌4,8のクリアランスを阻害する知られています。食細胞と肺の恒常性の最初の行の擁護者としての役割のほか AMs 知られている T を引き出すことで抗原提示細胞細胞免疫9、として機能する鼻腔内ワクチン10の効果を増強し、肺移植11,12後肺制限自己免疫に影響を与えます。午前の機能の欠乏は、遺伝子の突然変異、悪性腫瘍や肺界面活性剤13,14のクリアランスを損なう感染から生じる条件 (PAP)、肺胞蛋白症にリンクされています。AMs の移植は今 PAP 15,16の治療のための治療法として検討されています。
AMs は、胚形成の間に由来して循環白血球2,17に置き換えることがなく一生肺に固執するに知られています。午前売り上げ高のさまざまなレベルがインフルエンザ ウイルス4、破壊的照射18、内毒素への暴露によって感染を含む特定の臨床状況で報告されている午前売り上げ高は恒常性の肺に、19、そして高齢者20。AMs は、自己を介して悪性増殖17,21, 更新すると考えられているが、いくつかの最近の研究は、単球が血管肺マクロファージ22,23の下の人口に上昇を与えることができることを主張これらの新たに変えられた肺のマクロファージの機能がの実験条件はまだ肺疾患定義しなければなりません。肺炎症シグナルと免疫制御機械間の平衡を維持しようとすると、潜在的に興味深い地域がさらに午前活性化のコンテキストで刺激の閾値を理解することです。
免疫制御の損失をもたらす生理学的または病理学的変化は、さまざまな臨床設定 (例えば、呼吸器感染症、炎症性肺疾患、肺線維化疾患) の評価に重要です。それにもかかわらず、AMs はますますインジケーターまたは肺健康11,24のも決定要因として認識されます。収穫、特性評価、および人間および前臨床マウスモデルから維持する AMs 統一プロトコルは現在、ありません。午前前駆体と表現型のコンセンサスの欠如と詳細な方法の不在は、肺の健康および病気の午前の解読の役割の主要な障害をされていた。次のプロトコルでは、決定的な識別、分離、および体外で文化大きく午前動作の理解を進めるし、AM 向けの診断および治療の調査を促進する戦略を提供しています。

<table><tbody><tr><td>Non-enzymatic cell dissociating solution</td><td>Millipore-Sigma</td><td>C5789</td><td></td></tr><tr><td>Puralube Vet Ointment</td><td>Dechra</td><td>620300</td><td></td></tr><tr><td>22G Catheter&amp;nbsp;</td><td>Terumo Medical Products</td><td>SR-OX2225CA</td><td></td></tr><tr><td>4-0 Non-absorbable silk braided suture&amp;nbsp;</td><td>Kent Scientific</td><td>SUT-15-2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>Mouse Fc block&amp;nbsp;</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>Lysis buffer (PureLink RNA Kit)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>12183018A</td><td></td></tr><tr><td>b-Mercaptoethanol&amp;nbsp;</td><td>Millipore-Sigma</td><td>M6250&amp;nbsp;</td><td></td></tr><tr><td>FACSAria II cell sorter&amp;nbsp;</td><td>BD Biosciences</td><td>644832</td><td></td></tr><tr><td>Ketamine&amp;nbsp; (Ketathesia)</td><td>Henry Schein</td><td>56344</td><td></td></tr><tr><td>Xylazine&amp;nbsp; (AnaSed)</td><td>Akorn</td><td>139-236</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CM</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CM</td><td></td></tr><tr><td>Liberase TL&amp;nbsp;</td><td>Millipore-Sigma</td><td>5401020001</td><td></td></tr><tr><td>DNase I</td><td>Millipore-Sigma</td><td>AMPD1-1KT</td><td></td></tr><tr><td>100&amp;mu;m cell strainer&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Human Fc block</td><td>BD Biosciences</td><td>564220</td><td></td></tr><tr><td>EDTA</td><td>Corning</td><td>46-034-CI</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AMQAX1000</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15250061</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150H</td><td></td></tr><tr><td>L-929 cell line</td><td>American Type Culture Collection</td><td>ATCC,&amp;nbsp;CCL-1</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Corning</td><td>25-000-CI</td><td></td></tr><tr><td>T25 Tissue culture flask</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>156367</td><td></td></tr><tr><td>60 mm culture dish&amp;nbsp;</td><td>Millipore-Sigma</td><td>CLS3261</td><td></td></tr><tr><td>15 mL Conical tube&amp;nbsp;</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>50 mL Conical tube&amp;nbsp;</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>LSRFortessa cell analyzer</td><td>BD Biosciences</td><td>657669</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo</td><td>v10.4</td><td>Analysis Software</td></tr><tr><td>Anti-CD45 (Mouse)</td><td>Biolegend</td><td>147709</td><td>Clone I3/2.3, FITC conjugated</td></tr><tr><td>Anti-CD11b (Mouse)</td><td>Biolegend</td><td>101228</td><td>Clone M1/70, PerCP/Cy5.5 conjugated</td></tr><tr><td>Anti-CD11c (Mouse)</td><td>BD Biosciences</td><td>565452</td><td>Clone N418, BV 421 conjugated</td></tr><tr><td>Anti-I-Ab (Mouse)</td><td>Biolegend</td><td>116420</td><td>Clone AF6-120.1, PE/Cy7 conjugated</td></tr><tr><td>Anti-Siglec-F (Mouse)</td><td>BD Biosciences</td><td>562757</td><td>Clone E50-2440, PE-CF594 conjugated</td></tr><tr><td>Anti-Siglec-H (Mouse)</td><td>Biolegend</td><td>129605</td><td>Clone 551, PE conjugated</td></tr><tr><td>Anti-F4/80 (Mouse)</td><td>Biolegend</td><td>123118</td><td>Clone BM8, APC/Cy7 conjugated</td></tr><tr><td>Anti-Ly-6C (Mouse)</td><td>Biolegend</td><td>128035</td><td>Clone HK1.4, BV605 conjugated</td></tr><tr><td>Anti-CD64 (Mouse)</td><td>Biolegend</td><td>139311</td><td>Clone X54-5/7.1, BV711 conjugated</td></tr><tr><td>Anti-CD24 (Mouse)</td><td>BD Biosciences</td><td>563115</td><td>Clone M1/69, BV510 conjugated</td></tr><tr><td>Anti-CD103 (Mouse)</td><td>BD Biosciences</td><td>745305</td><td>Clone OX-62, BV650 conjugated</td></tr><tr><td>Anti-CD317 (Mouse)</td><td>Biolegend</td><td>127015</td><td>Clone 927, APC conjugated</td></tr><tr><td>Anti-CXCR1 (Mouse)</td><td>Biolegend</td><td>149029</td><td>Clone SA011F11, BV785 conjugated</td></tr><tr><td>Anti-CD45 (Human)</td><td>Biolegend</td><td>304017</td><td>Clone HI30, AF488 conjugated</td></tr><tr><td>Anti-CD11b (Human)</td><td>Biolegend</td><td>101216</td><td>Clone M1/70, PE/Cy7 conjugated</td></tr><tr><td>Anti-HLA-DR (Human)</td><td>Biolegend</td><td>307618</td><td>Clone L243, APC/Cy7 conjugated</td></tr><tr><td>Anti-CD169 (Human)</td><td>Biolegend</td><td>346008</td><td>Clone 7-239, APC conjugated</td></tr><tr><td>Anti-CD206 (Human)</td><td>Biolegend</td><td>321106</td><td>Clone 15-2, PE conjugated</td></tr><tr><td>Anti-CD163 (Human)</td><td>Biolegend</td><td>333612</td><td>Clone GHI/61, BV421 conjugated</td></tr></tbody></table>

isolation and in vitro culture of murine and human alveolar macrophages

肺胞マクロファージは、肺居住者、最終分化マクロファージ出生前原産です。肺胞マクロファージは、寿命が長く、肺開発機能とその肺局所応答感染症や炎症に重要な役割でユニークです。日には、識別、分離、および人間およびマウスから肺胞マクロファージの処理の統一的手法は存在しません。このようなメソッドは、さまざまな実験的設定のこれらの重要な自然免疫系の細胞に関する研究に必要です。あらゆる研究室で簡単に採用できる、ここで説明する方法は、気管支肺胞洗浄液や肺組織から肺胞マクロファージを採取し、体外を維持する簡素化されたアプローチです。肺胞マクロファージは、肺胞に付着性のセルとして主に発生する、ためこのメソッドの焦点は収穫と同定する前にそれらを外れです。肺は高度に血管の臓器、骨髄性とリンパ性の起源の様々 な細胞タイプに生息する、対話、および肺微小環境によって影響を受けています。ここで説明されている表面のマーカーのセットを使用して、研究者はことができます簡単かつ明確に他の白血球から肺胞マクロファージを区別し、下位アプリケーションにそれらを浄化します。ここを開発した培養法両方の人間をサポートするマウスの体外発育の肺胞マクロファージと分子・細胞の研究と互換性が。

マウス AMs を識別するためにフローのフローサイトメトリーによるアプローチは、図 1に示すです。これは、表面マーカー他肺居住者又は肺に浸潤食から AMs を区別するに必要な最小セットの分析が含まれています。差分解析は、間質性マクロファージ、樹状細胞、好中球、単球、肺に発生する単球由来の肺マクロファージから AMs を識別するた?...

Watch this Scientific Journal Video about Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages at JoVE.com

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

離とマウスとヒトの肺胞マクロファージの体外培養

この通信は、人間から肺胞大食細胞の分離と培養の方法論や実験用マウスのモデルについて説明します。

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

isolation-vitro-culture-murine-human-alveolar

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JoVE Journal

Immunology and Infection

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

23.5K ビュー.  St. Joseph's Hospital and Medical Center. この通信は、人間から肺胞大食細胞の分離と培養の方法論や実験用マウスのモデルについて説明します。

ビデオ: Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

免疫・感染学

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

マウス肺胞マクロファージを採取し、無水物ナノ粒子により誘導される細胞活性化を評価する

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工学

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

ダブル勾配遠心、テフロンコーティングされた細胞培養バッグ中のマクロファージへの分化によるヒト単球の単離

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

共焦点顕微鏡による絶縁型肺胞マクロファージの抗真菌活性を評価する

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

ヒトおよびマウスの心筋標本からのマクロファージサブセットおよび間質細胞の分離

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

マウス由来骨髄由来マクロファージの単離と培養

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

マウス洞房・房室結節からの常在型心臓マクロファージの単離と培養

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...