Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

研究は、シンガポール（ACRFのTier1-RG40 / 13およびTier2の-MOE2013-T2-2-038）、教育省によってサポートされています。原稿がObrizusコミュニケーションズからのエイミー・サリバンが編集しました。

全てのマウスを交配させ、特定の病原体を含まない条件下で維持し、すべてのマウスプロトコルは、施設内動物管理使用委員会のガイドラインに従って行われています。 緩衝液および試薬の調製<ol><li> 、55μM2-メルカプトエタノールを完全ロズウェルパーク記念研究所（RPMI）培地（10％熱不活性化ウシ胎児血清（FBS）、2mM L-グルタミン、ペニシリン（100 IU / ml）を/ストレプトマイシン（100μg/ ml）を準備します）。 </li><li>これとは別に、20倍の平衡塩類溶液（BSS）ストック1およびストック2を準備します。 <ol><li> 20倍BSSストック1（1 Lの滅菌水で111 mMのブドウ糖、8.8 mMリン酸カリウム、26.7 mMのリン酸ナトリウム二塩基性）を準備し、最終容量調整前の20倍BSSストック1に40ミリリットルの0.5％フェノールレッドを追加します。 4℃で保存する前に、0.2μmのフィルターを用いて滅菌濾過します。 </li><li> 20X BSSストック2（25.8 mMの塩化カルシウム二水和物、107 mMの塩化カリウム、2.73 MナトリウムCHを準備loride、19.6 mMの塩化マグネシウム六水和物、1 Lの滅菌水で16.6 mMの硫酸マグネシウム）。 4℃で保存する前に、0.2μmのフィルターを用いて滅菌濾過します。 </li><li> 400ミリリットル滅菌水それぞれに、別々に、50ミリリットルBSSストック1と50ミリリットルBSSストック2を希釈し、実験的な使用のために1X BSSを準備します。 、両方の希釈液を組み合わせてpHを7に調整し、20ミリリットルのFBS（2％）を追加します。 0.2μmのフィルターを用いて滅菌水と滅菌フィルターを使用して1 Lまでトップ。 注：濃厚BSSストックの混合が直接析出につながる可能性があるため、BSSストック溶液を別途準備する必要があります。 </li></ol></li><li>赤血球（RBC）溶解バッファー<ol><li> RBC溶解緩衝液を調製するために、使用前に1部株式トリス塩基（滅菌水中の130mMのトリス（ヒドロキシメチル）アミノメタン、pHは7.65）に9部ストック塩化アンモニウム（滅菌水中の155 mMの塩化アンモニウム）を混合します。 注：4℃で塩化アンモニウムおよびトリスベースのストア滅菌ストック溶液。溶解bを準備しますRBCの効率的な溶解を確実にするために、新鮮なuffer。 </li></ol></li></ol>脾臓またはリンパ節からリンパ球懸濁液の2世代注：マウスの安楽死の前に、実験に必要な全ての試薬および装置を準備して、高い細胞生存率を維持するために、できるだけ早くリンパ球の単一細胞懸濁液を生成することが重要です。 <ol><li>頸椎脱臼またはCO 2窒息により実験マウスを安楽死させます。 注：これ以降のステップで、すべての実験手順は、無菌的に行うべきです。 </li><li>任意の切開を行う前に、70％エタノールにマウス全体を浸し。無菌的に8脾臓およびリンパ節を削除し、（ステップ1.2.3から）5ミリリットルの氷冷RPMI / FBS（2％FBSを含むRPMI）やBSS / FBSを含む別の15ミリリットルチューブに配置します。 注：BSSは、効率的なRBC溶解を防止するため、脾細胞懸濁液を調製するためのRPMIを使用しAFTE BSSに切り替えますRBC溶解をrは。 </li><li> 2ミリリットルの氷冷RPMI / FBSまたはBSS / FBSを含むペトリ皿に無菌100μmのセルストレーナーメッシュの2枚の間に臓器（複数可）を置き、脾臓またはリンパ節からの単一細胞懸濁液を生成するには。それは非常に細かい部分に引き裂かれるまで1mlシリンジのプランジャーを用いて、器官（複数可）をマッシュ。 </li><li> 15ミリリットルチューブに細胞懸濁液を移し、セルストレーナーを氷冷RPMI / FBSまたはBSS / FBSでメッシュ洗います。 、残りの細胞懸濁液を収集し、同じ15 mlチューブに追加し、4℃で5分間、453×gでスピンダウン。上清を取り除きます。 注：後続のステップでRBC溶解緩衝液または培地を添加する前に、指でチューブをフリックすることによりペレット化した細胞を再懸濁します。 </li><li>細胞懸濁液の遠心分離中に室温（RT）RBC溶解バッファーを準備し（ステップ1.3を参照してください。）。細胞をペレット化し、上清を除去した後、すべての10 8個の細胞1mlのRBC溶解緩衝液を用いて細胞を再懸濁します。 IncuBATE 3-4分間室温で溶解反応。 </li><li> 14ミリリットルの氷冷BSS / FBSを停止RBC溶解し、4℃で5分間、453×gでスピンダウン。 </li></ol> B及びT細胞の3精製<ol><li> B細胞の精製<ol><li>血球計数器を用いて細胞を数えます。 300μlのBSS / FBSで最大10 8脾臓細胞を再懸濁し、50μlの抗CD43に磁性マイクロビーズ9,10を追加します。 、死んだ細胞を除去30μlのアネキシンV磁気ビーズを追加します。 30分間、4℃の冷蔵庫中の細胞懸濁液をインキュベートします。 </li></ol></li><li> T細胞の精製<ol><li>血球計数器を用いて細胞を数えます。再懸濁し、標的細胞集団に応じて非T細胞枯渇抗体カクテルで10 8細胞CD19、B220、GR-1、TCR-γδ、CD49b、CD11cの、のCD11b、TER119およびCD4またはCD8に対する（ビオチン化抗体まで/ FBS 4,5200μlのBSSに200：）精製され、1に希釈しました。中の細胞懸濁液をインキュベート15分間、4℃の冷蔵庫。 </li><li>インキュベーション後、細胞を洗浄し、4℃で5分間、453×gでスピンダウンする10ミリリットルBSS / FBSを追加します。上清を除去し、30μlのストレプトアビジンマイクロビーズおよび15μlのアネキシンV磁気ビーズを165μlのBSS / FBS中で細胞を再懸濁します。 30分間、4℃の冷蔵庫中の細胞懸濁液をインキュベートします。 注：、あっても磁性マイクロビーズで標識することを確認し、15分間マイクロビーズを用いて細胞をインキュベートし、その後、15ミリリットルチューブをタッピングにより穏やかに細胞懸濁液を混合し、ステップ3.1.1の間に、別の15分間インキュベートします。または3.2.2。 </li></ol></li><li>細胞精製のための分離カラムの調製<ol><li>細胞のマイクロビーズ標識の中に未使用の分離カラムを準備する（ステップ3.1.1。または3.2.2。）。 RTへ（FBSなし）プリ暖かいBSSと無菌的にカラムを洗浄し、平衡化するために2ミリリットルを使用しています。 BSSで平衡化した後、洗浄し、カラムを乾燥させてはなりません。 注：私たちはLS cを使用しますolumn代わりに推奨されるLDの列の、その再利用性に（ステップ3.4を参照してください。）。 </li><li>磁気ビーズで標識した後、細胞を洗浄し、4℃で5分間、453×gでスピンダウンする14ミリリットルBSS / FBSを追加します。上清を除去し、1-3で細胞ミリリットルRT BSSを再サスペンド。 </li><li>精製のプロセスの間に流量を減らすために、カラムの先端に滅菌21 Gの針を取り付けます。平衡化したカラムに細胞懸濁液をロードし、精製された標的細胞を含有するフロースルーを集めます。 注：ロード中にカラムに気泡を導入することは避けてください。 </li><li> 1ミリリットルBSS / FBSで一回カラムを洗浄し、精製された標的細胞を含有するフロースルーを集めます。再び通る流れでカラムをリロードします。同じ15ミリリットルチューブ内の第二の負荷後のフロースルーを収集します。 </li><li> 1ミリリットルBSS / FBSでカラムを3回洗浄し、精製された標的細胞を含有するフロースルーを集めます。その後、目に5ミリリットルBSS / FBSを追加電子カラムと、プランジャーと、新しい15ミリリットルチューブにカラムから磁気標識された細胞を洗い流します。 </li><li>精製されたBまたはT細胞4,5の表面抗原に結合する抗体を用いたフローサイトメトリーによって収集された細胞の純度を確認してください。 </li></ol></li><li>分離カラムを使用して再 注：LSカラムは、浄化効率に影響を与えることなく、4回まで再利用することができます。 <ol><li>プランジャーを使用して上から水を5mlの蒸留で5 mlのリン酸緩衝生理食塩水（PBS）で3回カラムを3回洗浄します。 </li><li> 5ミリリットル70％エタノールでカラムを洗浄し、カラムに錆の蓄積を防ぐために空気タップを使用して広範囲にカラムを乾燥。 </li><li>個別の精製実験のために使用されるLSカラムを準備するには、シリンジアダプタを使用して、5ミリリットル、70％エタノールで下から上カラムを洗浄。次に、5ミリリットル一度列の先頭からPBSに続いて5ミリリットル滅菌PBSで2回ボトムアップからカラムを洗浄。 2を追加します。ミリリットルRT BSSは、カラムを平衡化した後、標識された細胞でカラムをロードするに進みます。 </li></ol></li></ol> 4. CFSE標識および刺激注：精製細胞は、in vitroでの様々なおよびインビボ機能アッセイにかけることができます。ここでは、APCは5のT細胞刺激能を決定するために、精製されたT細胞を使用します。 <ol><li> 37°Cの前CFSEをロードする前暖かい標識溶液（PBS中0.1％FBS）。 注：PBS中のFBSの低い割合を使用すると、CFSEのロード中に細胞死を減少させ、遠心分離中の細胞損失を最小限に抑えることができます。しかし、あまりにも多くのFBSはCFSEのロードを妨げる可能性があります。 </li><li>標識溶液で2回精製した細胞を洗浄し、15mlチューブ中の予め温めた標識溶液中で2×10 7細胞/ mlで再懸濁しました。 </li><li>予め温めておいた標識溶液中で：（5 mMのCFSEストック溶液の500倍希釈）10μMのCFSE溶液を調製します。 CFSEソリューションべきたて最適なラベリングを達成するたびに調製すること。 </li><li> CFSEで細胞をロードするには、15 mlチューブで1部10μMのCFSE溶液に1部の細胞懸濁液を追加し、37℃で10分間、暗所でインキュベートします。 5μMのCFSEの最終濃度は、1×10 7細胞/ mlを標識するために使用されます。 </li><li> CFSEのロード中に細胞の均質な混合物を確実にするためにチューブを2分ごとに反転します。 </li><li>反応を停止するには、氷のように冷たい完全RPMI培地の複数のボリュームを追加し、4℃で5分間、453×gでスピンダウン。成功したCFSEのロード時に、細胞ペレットを、黄色がかった表示されます。 </li><li>ウォッシュCFSEは氷のように冷たい完全RPMI培地で細胞を1より多くの時間をロードされ、in vitroでの培養のためにまたはインビボ刺激に使用する前に、4℃で5分間、453×gでスピンダウン。 </li></ol> 5. in vitro刺激<ol><li>使用直前に2倍の刺激のストック溶液（2×刺激原液）を調製10となるよう2X刺激原液0μlを96ウェルプレート中のウェルあたり200μlの最終容積に細胞100μlを添加することができます。 </li><li> （B細胞またはT細胞のためのCD3およびCD28のためにIgMまたはCD40）の刺激でコーティングされたプレートが必要な場合は、4℃で一晩PBSで刺激し、プレコート培養プレートを希釈します。代替的に、培養プレートは、実験の当日に1時間37℃でコーティングすることができます。 （プレートは任意の時点で乾燥させないでください）PBSで2回コーティングしたプレートを洗浄します。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> B細胞について </td><td></td></tr><tr><td> 刺激 </td><td> 最終濃度 </td></tr><tr><td> F（ab &#39;）2ヤギ抗マウスIgM </td><td> 0.6から2.4 / mlの</td></tr><tr><td>抗マウスCD40モノクローナル抗体</td><td> 0.5 / mlの</td></tr><tr><td>組み換え体マウスIL-4 </td><td> 25 U / mlの</td></tr><tr><td>リポ多糖類</td><td rowspan="2"> 0.1 / mlの</td></tr><tr><td> E.から（LPS） 大腸菌血清型055：B5 </td></tr><tr><td></td><td></td></tr><tr><td> T細胞のための </td><td></td></tr><tr><td> 刺激 </td><td> 最終濃度 </td></tr><tr><td>抗CD3（プレートコーティングされました） </td><td> 2-10 / mlの</td></tr><tr><td> （50μL/ウェルコーティング用） </td><td></td></tr><tr><td>抗CD28（プレートコーティングされました） </td><td> 2 / mlの</td></tr><tr><td>組換えIL-2 </td><td> 40 U / mlの</td></tr><tr><td> PDBu（ホルボールエステル） </td><td> 5-50 ng / mlで</td></tr><tr><td> A23187（カルシウムイオノフォア） </td><td> 250 ng / mlで</td></tr></tbody></table> 表1： インビトロ培養中のリンパ球を刺激するために使用される刺激の濃度。<ol start="3"><li> CFSE標識B細胞の場合は、3×10 5細胞/ウェルで96ウェルの72時間平底プレート三連で完全RPMI培地と培養3×10 6細胞/ mlに再懸濁します。 </li><li> CFSE標識T細胞を、0.5~3×10 5細胞で三重で完全RPMI培地及び培養中の0.5-3×10 6細胞/ mlに再懸濁/ウェルで48または72時間、96ウェル丸底プレート。 </li></ol> 6. インビボ刺激<ol><li> in vivoでの刺激、各MHC適合レシピエントマウスに養子移入（200μlのPBS中の静脈内（IV））マウスあたり4×10 6 CFSE標識T細胞について。 注：このプロトコルでは、CFSE標識T細胞は、養子意志家など脾臓およびリンパ節などのリンパ器官へのこれらの細胞のように尾静脈または眼窩後注射を使用して転送することができます。 </li><li> 1日後に抗原とレシピエントマウスに挑戦。 注意：OVA特異的T細胞受容体（TCR） - トランスジェニックT細胞を養子レシピエントマウスに移したため、この例では、我々は、抗原としてオボアルブミン（OVAタンパク質を50μg/マウス）を使用します。滅菌PBSでOVAタンパク質を準備し、各レシピエントマウス5に、皮下注射（SI）を経由して、OVAタンパク質/ PBSまたはPBS対照の100μLを注入します。 </li><li>収穫し、3日間OVAタンパク質またはPBSで免疫した後、レシピエントマウスのリンパ器官（リンパ節および脾臓）からの単一細胞懸濁液を生成します。腸間膜、膝窩、鼠径部、腰部、および尾部リンパ節を含んで腋窩、上腕および表在性頸部リンパ節を含んで近位リンパ節（PLN）、）および遠位リンパ節（DLN）への個別のリンパ節、。 T細胞の増殖を確認するために、適切なFACS抗体を用いて染色細胞。 注：このCFSE細胞追跡実験において、PBSを注射した対照マウスからのCFSE標識T細胞は非 - ジのベースライン蛍光を確立します細胞をviding。増殖の細胞分裂は、抗原刺激、CFSE標識細胞は、蛍光ピーク12,13を測定することによって可視化されます。 PBSコントロールで、増殖の細胞分裂の数は、CFSE標識T細胞は、12,13を決定することができます。 </li><li>サンプル間の細胞分裂またはピーク数を比較することによって、CFSE標識細胞増殖データを分析した （ 図1及び2）。 注：例えば、養子OVA抗原を受けたレシピエントマウスに移しCFSE標識、OVA特異的T細胞は、PBS注射コントロールマウス5,12に比べて活発な増殖を受けます。また、ホーキンスら14により示されるようにCFSE細胞増殖データを分析するための多くの方法が存在することを指摘することは注目に値します。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171 (1), 131-137 (1994).</li><li>Cabatingan, M. S., Schmidt, M. R., Sen, R., Woodland, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na&#239;ve+B+lymphocytes+undergo+homeostatic+proliferation+in+response+to+B+cell+deficit.">Na&#239;ve B lymphocytes undergo homeostatic proliferation in response to B cell deficit.</a> J. Immunol. 169 (12), 6795-6805 (2002).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Th17+differentiation+of+na&#239;ve+CD4+lymphocytes.">Isolation and Th17 differentiation of na&#239;ve CD4 lymphocytes.</a> J. Vis. Exp. (79), e50765 (2013).</li><li>Su, 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=Ezh2+controls+B+cell+development+through+histone+h3+methylation+and+Igh+rearrangement.">Ezh2 controls B cell development through histone h3 methylation and Igh rearrangement.</a> Nat. Immunol. 4 (2), 124-131 (2003).</li><li>Mecklenbr&#228;uker, I., Saijo, K., Zheng, N., Leitges, M., Tarakhovsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C&#948;+controls+self-antigen-induced+B-cell+tolerance.">Protein kinase C&#948; controls self-antigen-induced B-cell tolerance.</a> Nature. 416 (6883), 860-865 (2002).</li><li>Rush, J. S., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+activated+via+CD40+and+IL-4+undergo+a+division+burst+but+require+continued+stimulation+to+maintain+division,+survival+and+differentiation.">B cells activated via CD40 and IL-4 undergo a division burst but require continued stimulation to maintain division, survival and differentiation.</a> Eur. J. Immunol. 31 (4), 1150-1159 (2001).</li><li>Quah, B. J. C., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat. Protoc. 2 (9), 2049-2056 (2007).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> J. Immunol. Methods. 379 (1-2), 1-14 (2012).</li><li>Hawkins, E. D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nat. Protoc. 2 (9), 2057-2067 (2007).</li><li>Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation:+Terminology+and+practical+considerations.">Cell separation: Terminology and practical considerations.</a> J. Tissue Eng. 4 (1), 1-14 (2013).</li></ol>

The authors have nothing to disclose.

このプロトコルでは、我々は、リンパ器官からのリンパ球を精製するための手順を示します。磁気ビーズ選別を用いた細胞の精製は、実行可能な、高度に精製された標的細胞をもたらす迅速かつ簡単な方法です。 プロトコル内の重要なステップ 細胞生存率および細胞収量 in vitroで造血系細胞の生存率を維持す?...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

リンパ球の磁性細胞精製は、ユーザが比較的短い時間で、標的細胞集団を精製することができます。我々の枯渇のプロトコルを用いて、精製した後、94.2％（に72.8％（前の精製）から（組換え活性化遺伝子1（RAG-1）欠損マウスにおけるOT-I）CD8 T細胞の割合を増加させることができました。 図1A）4,5。これらの精製されたリンパ球は、その後、リン?...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

単離とマウスリンパ球の活性化

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

21.0K ビュー.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

ビデオ: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

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

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

B細胞のCD40 -活性化のモデルマウス

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

免疫・感染学

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

マウス単球由来樹状細胞とその後続の in Vitro活性化腫瘍免疫複合体との隔離のプロトコル

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

がん研究

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4 +ミルテニーMACS精製を使用してマウスのリンパ節からT細胞の分離

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

マウス生殖管粘膜からのリンパ球の単離

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

マウス真皮リンパ管からの内皮細胞の単離

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

著者スポットライト:マウス真皮リンパ内皮細胞の磁気ビーズベースの単離

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

マウス真皮リンパ管内皮細胞の単離

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...