Name: efficient isolation protocol for b and t lymphocytes from human palatine tonsils
Uploaded: 2015-11-16T14:00:00.000+00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

プロトコルは、ヒト患者材料からのリンパ球様細胞の単離を記載し、したがって、倫理的な承認を必要とします。本研究で行われた作業は、ウプサラ倫理審査委員会（DNR。387分の2013）によって付与されました。 ヒトパラティーノ扁桃腺から単核細胞（MNCを）の1の単離注意：血液、組織または体液のようなヒト由来の全てのシールドの材料は、感染した可能性材料とみなされるべきです。したがって、ヒト組織を処理するための推奨されるバイオセーフティ慣行に従うべきです。 注：汚染を避けるために、全ての溶液及び細胞培養装置は、無菌でなければなりません。全ての緩衝液および溶液は、事前に冷却し、氷上に保持する必要があります。扁桃組織および単離した細胞を保持し、氷上で処理する必要があります。 <ol><li>扁桃摘出やへんとう切除（図2）を受けた患者からの新鮮な扁桃腺を取得します。組織clumプランジ氷冷滅菌ハンクス平衡塩溶液を充填した50 ml遠心管（HBSS）、5％ウシ胎児血清（FBS）を補充し、10 mMのグルタミン、0.05 mg / mlのゲンタマイシンおよび1％抗生物質 - 抗真菌ミックス（ペニシリン、にPSストレプトマイシン及びアンホテリシンB）。 </li><li>氷の上に常時水没扁桃組織サンプルを用いてチューブを保管してください。できるだけ早く扁桃腺を処理しようとせず、遅くとも3時間よりも外科的除去後。 </li><li>氷上で60ミリメートルのプラスチック細胞培養プレートに扁桃腺を置き、HBSSで湿らせたティッシュを維持します。扁桃表面からのきれいなピンセットで目に見える血液凝固、脂肪および結合組織を除去します。 </li><li> 5ミリリットルのHBSSを含む新しい60ミリメートルの細胞培養プレートを使用してください。はさみおよび/またはメスの無菌のペアを使用して3〜10ミリメートルの断片に扁桃組織の塊をカットします。 </li><li> HBSS 10mlのを含む新しい60ミリメートルの細胞培養プレートを準備し、HBSS溶液中に100μmのプラスチック製セルストレーナーを配置します。 </li><li>ディス転送滅菌ピンセットを用いて細胞ストレーナーに扁桃腺フラグメントをected。プラスチックシリンジのプランジャの端を使用して、スムーズにセルストレーナーを介して組織片を絞ります。扁桃腺断片が完全にHBSS中に浸漬されていることを確認します。 </li><li>組織が残存して細胞ストレーナーを廃棄し、50mlプラスチック遠心管に60 mmの細胞培養プレートから得られた細胞懸濁液を移します。ステップ1.8および1.9を進めながら、氷上でチューブを残します。 </li><li>大きさが2cm（ 図2）よりも大きい扁桃腺の場合には、ストレーナメッシュの目詰まりを回避するために、2つのセルストレーナーで扁桃腺断片を絞ります。 </li><li>細胞懸濁液を35 mlの最終容積を得ます。 注：このステップでは、凍結保存（セクション2を参照）の細胞懸濁液を調製することが可能です。 </li><li>新しい50ml遠心管にフィコール等密度勾配溶液10mlを加えます。静か細胞suspensをオーバーレイイオン密度勾配液の上に（ステップ1.9）。細胞懸濁液および密度勾配溶液の混合を避けます。 注：密度勾配溶液を室温まで加温しなければなりません。 </li><li> RTで20分間、700×gで、スイングアウトローター中で細胞懸濁液を遠心。勾配が中断される可能性があり、高速ブレーキとして遠心分離機のブレーキ機能をアクティブにしないでください。また、遠心分離器で利用可能な最低の加速関数を使用します。 注：この遠心分離工程の後、チューブの底に沈殿物として界面でふわふわした白色層、および赤血球（RBC）、線維芽細胞および細胞残屑などのMNCを観察します。 </li><li>慎重に10ミリリットルピペットを用いて、多国籍企業層を収集します。新しい50ミリリットルの遠心管に細胞懸濁液を置きます。 </li><li>細胞懸濁液に氷冷PBS 25 mlを加え、4℃でスイングアウトローター中で5分間300×gでチューブをスピン。 </li><li> 25ミリリットルピペットを用いて上清を削除細胞ペレットの洗浄工程をさらに2回繰り返します。最後のPBS 15mlに細胞ペレットを再懸濁します。 注：最後の洗浄工程の後に、（セクション2を参照）の細胞懸濁液を凍結保存することが可能です。 </li><li>血球計数器細胞計数室に10μlの細胞懸濁液を適用し、顕微鏡下で細胞数をカウントします。後続の磁気ビーズ分離に15 mlの遠心管中の細胞の適切な量（例えば、2-3×10 7）を分注します。ステップ3.2まで氷上にこのアリコートを保管してください。 </li></ol>扁桃細胞の凍結保存2. <ol><li>凍結培地（90％FBSおよび10％DMSO）を準備し、氷の上に保管してください。長期保存のために-20℃で凍結メディアを保持します。 </li><li>血球計数器の細胞計数室（ステップ1.15）を用いて細胞の総数を決定します。希望の凍結細胞密度に応じて凍結媒体の必要量を計算する（例えば、10 7細胞凍結培地のS / mlで）。 </li><li> 5分間300×gで細胞懸濁液を遠心。細胞ペレットを乱すことなく上清を除去し、（ステップ2.1から）を氷冷凍結培地中で細胞ペレットを再懸濁します。 </li><li>液体窒素中で長期保存のために設計され、滅菌バイアル中に細胞懸濁液1mlのアリコートを分配します。イソプロパノール室でバイアルを凍結し、-80°のCO / Nでそれらを格納します。長期保存のために貯蔵タンクまたは-140℃の細胞冷凍庫を含む液体窒素中にバイアルを転送します。 </li><li>凍結細胞とのバイアルを解凍し、37℃の水浴中で急速にそれらを暖めます。解凍した直後と、15mlの遠心管中で（サプリメント、ステップ1.1）、予め温めておいたHBSS 10mlの中に細胞懸濁液を分散させます。 、5分間300×gでチューブをスピンHBSSを除去し、（サプリメントと、ステップ1.1）HBSS中の所望の細胞密度で細胞ペレットを再懸濁。 注意：多国籍企業AFの実行可能性をター解凍は死細胞の存在は、精製されたBおよびTリンパ球の最終収率が低下するので、重要です。 注：細胞単離の効率を低下させる、細胞生存率が80％、次に我々の経験によります。 </li></ol>扁桃多国籍企業からのTリンパ球の人口の3ポジティブ選択注1：このプロトコルは、CD3抗体に結合された磁気ビーズを使用して、扁桃多国籍企業からのヒトCD3 + Tリンパ球の正の選択に基づいています。これは、新鮮な（セクション1）又は冷凍（セクション2）多国籍企業から、このセクションを開始することが可能となります。 注2：3×10 7の多国籍企業から開始します。分離カラムが詰まることがあり、これは、分離効率が低下するので、この細胞数を超えないようにしてください。より多くの細胞が処理される場合は、より大きな列を使用します。このプロトコルで使用されるボリュームは、実験的に細胞たちの数のために最適化されています私たちの実験で編。 <ol><li>分離緩衝液[PBS（pH7.2）中、0.5％ウシ血清アルブミン（BSA）および2mM EDTA]を準備します。 4℃で0.45μmフィルターとストアを介して分離バッファをフィルタリングします。 </li><li> 4℃で5分間、300×gでのMNC懸濁液（ステップ1.14）をスピン。氷冷分離緩衝液の240μlの（10 7個の細胞あたり80μl）をで得られた細胞ペレットを再懸濁します。無菌の2mlチューブに細胞懸濁液を転送します。 </li><li>細胞溶液にCD3磁気抗体20μlのを追加します。懸濁液中の細胞を維持するために連続的に穏やかに混合しながら4℃で1時間、チューブをインキュベートします。 </li><li>細胞を洗浄し、5 mlの氷冷分離緩衝液を含む15 mlのチューブに細胞懸濁液の全てを転送します。 </li><li>スイングアウトローターで4℃で10分間300×gでチューブを遠心。 </li><li>一方、磁気分離と列を設定します。スタンドに磁気分離器を取り付け、SEPARで列を配置レータ。氷冷分離緩衝液500μlを適用することにより、カラムを洗浄。フロースルーを捨てます。 </li><li>列の下に新しい15ミリリットルのコレクションチューブを置きます。氷の上にコレクションチューブを保管してください。 </li><li>ピペットで上清（ステップ3.5）を破棄し、ゆっくりと500μlの分離バッファで細胞ペレットを再懸濁します。予め洗浄したカラムの上に細胞懸濁液を適用し、それを介して、実行してみましょう。 注：細胞塊は、カラムを詰まらせるので、流量を減少させることができます。この問題を回避するには、列の上に40μmのプラスチック製セルストレーナーを配置します。このメッシュを通して細胞を渡すと、非常にこの方法の効率を改善します。 </li><li>分離緩衝液1.5mlの（10 7個の細胞のための500μl）を用いてカラムを4回洗浄します。次の洗浄工程を適用する前に、（液体が列で観察されるべきではない）空に列リザーバのを待ちます。 注：Bリンパ球画分としてみなさCD3非標識細胞を得る、そのままコレクションチューブに溶出しました。 </li><li>新しい15ミリリットルのコレクションチューブにセパレーターと場所から列を削除します。ピペットカラムに分離緩衝液2mlの。 Tリンパ球画分としてみなされる正に選択されたTリンパ球を、溶出カラム付属のプランジャーを使用して。 </li><li>必要に応じて精製された細胞（ステップ1.15）の数を決定します。細胞懸濁液は現在、下流の実験の準備ができています。 </li></ol>単離した扁桃BおよびTリンパ球の4フローサイトメトリー分析注意：パラホルムアルデヒドソリューションは、刺激物と疑われる発がん性物質です。適切な保護衣、手袋、眼/顔面用の保護具を着用します。 注1：このプロトコルは、（FACS）分析を蛍光活性化細胞選別によって分離されたB細胞およびT細胞を直接染色する方法が記載されています。この工程の主な目的は、単離された細胞集団のAFの純度を評価することですターCD3抗体磁気分離。 BおよびT細胞マーカー、フルオロフォア結合モノクローナル抗体CD20-FITCおよびCD2-APC確立し、この目的のために、使用されています。また、アイソタイプコントロール抗体を含めることが非常にCD2とCD20抗体（ステップ4.8を参照）の非特異的結合 &quot;背景&quot;を区別することをお勧めします。 注2：これは、染色時まで4℃で暗所で1％パラホルムアルデヒド（PFA）溶液に精製された細胞画分を維持することが可能である（例えば、次の日。）が。染色およびFACS分析の間の時間間隔がある場合にも同じ手順が適用可能です。どちらの場合も、細胞が染色前またはFACS分析にPBSA（0.2％BSAを含有するPBS）緩衝液で適切に洗浄する必要があることを覚えておいてください。 <ol><li>ステップ1.15からの多国籍企業の6×10 6個の細胞 （前の列に適用する）、ステップ3.9およびステップ3.10からのTリンパ球画分からのBリンパ球画分を取り出し。 </li><li>細胞をスピンスイングアウトローターを用いて4℃で5分間、300×gで懸濁液。 </li><li>ピペットで上清を除去し、氷冷PBSA 1mlで一度細胞ペレットを洗浄します。 4℃で5分間、300×gで細胞懸濁液をスピン。ピペットで上清を除去し、再懸濁 氷冷PBSAバッファー600μlの細胞。 </li><li> FACSチューブの底に100μlの細胞懸濁液を追加します。 </li><li>各チューブに、10％の熱不活性化ヒト血清を含む100μlのPBSを加え、よく混ぜ、氷上に〜RTで1分または20分間インキュベートします。 注：Bリンパ球は、Fc受容体を運びます。 Fcをブロックするために精製された細胞画分を、10％熱不活性化ヒト血清と共にインキュベートした受容体。ヒト血清を1時間56℃でインキュベーションすることにより熱不活性化です。 -20℃で凍結小分けとストアへの熱不活性化血清を分割します。 </li><li> SWで4℃で5分間、300×gで細胞懸濁液を遠心るアウトローター。 </li><li>ピペットで上清を除去し、1mlのPBSAで細胞ペレットを洗浄します。遠心分離（4℃で5分間、300×gで）を繰り返します。 </li><li>氷の上に各チューブに100μlのPBSAを追加します。製造業者の推奨に従って、フルオロフォアコンジュゲートモノクローナル抗体の適切な量を追加します（例えば、抗CD2020μlのおよび/ ​​またはPBSA100μlの抗CD2抗体を5μl）。注意：FACS分析のために十分な制御管を割り当てることを忘れないでください。このプロトコルでは、次のコントロールが含まれるべきである：1）細胞は、個々に、抗CD2および抗CD20抗体で染色し、抗CD2で染色した2）細胞と抗CD20アイソタイプ対照抗体単独で及び、3）細胞を添加していません抗体の。 </li><li>簡単に説明すると、チューブをボルテックスし、暗所で4℃で30分間インキュベートします。 </li><li> PBSAで2回洗浄します。サンプルに1％PFA溶液2mlを追加します。細胞は4でPFA溶液中に残ってみよう76; C暗所でのFACS分析時まで。 </li><li>直ちにFACS機でサンプルを実行する前に、（4℃で5分間、300×gで）をPBSAで細胞を2回洗浄します。 1mlのPBSと再懸濁サンプルを前に、フローサイトメーター上で分離し、光から保護し、4℃（または氷上）で保ちます。 </li><li>メーカーフローサイトメーターからのプロトコルに従って、FACSソーティングを行います。 </li></ol>単離した扁桃BおよびTリンパ球におけるアデノウイルスDNAの5 PCR検出注1：アデノウイルスヘキソン遺伝子プライマーはAdRJC1（5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;）及びAdRJC2（5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;）11です。これらのプライマーは、139塩基対のアンプリコンを生成します。ホスト18S rRNA遺伝子を、プライマーtp206（5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;）及びtp207（5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;）を用いて検出することができます。予想されるアンプリコンサイズは300 bpです。 注2：すべてのPCRランは、陰性対照（蒸留H 2 O）およびヒトアデノウイルス5型12に感染したヒトB細胞株（BJAB）から得られた陽性DNAコントロールを含んでいます。 <ol><li>シリカ膜ベースのカラム精製法を用いて、（少なくとも1×10 6個の細胞 、ステップ3.11から）のDNAを抽出します。フェノールとグアニジンイソチオシアネート液13を用いてRNAを抽出します。 </li><li> 20μlの総容量で、1×HF緩衝液を含む反応混合物にBおよびTリンパ球からの100ngのDNAを加えるデオキシヌクレオチド三リン酸混合物（のdNTP）の0.2mMの各プライマーの0.25μMと高忠実度DNAポリメラーゼの1 U。 </li><li>以下のサイクル条件でPCR増幅を実行します。30秒、72℃10秒、67℃（ヘキソン）または58℃（18S rRNAの）98℃の30サイクル、続いて98℃で30秒の変性、 30秒間。 PCR反応は、72℃で7分間の最終伸長工程で終了しました。 </li><li>得られたPCR amplifを分離1×TBE中の1.5％アガロースゲル上でication製品は、緩衝液及び核酸染色で染色することによって期待されるバンドを可視化します。 </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=Prevalence+and+quantitation+of+species+C+adenovirus+DNA+in+human+mucosal+lymphocytes.">Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes.</a> J Virol. 76, 10608-10616 (2002).</li><li>Perez, M. E., Billordo, L. A., Baz, P., Fainboim, L., Arana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+memory+B+cells+isolated+from+blood+and+tonsils+are+functionally+distinctive.">Human memory B cells isolated from blood and tonsils are functionally distinctive.</a> Immunol Cell Biol. 92, 882-887 (2014).</li><li>Cooper, R. J., Yeo, A. C., Bailey, A. S., Tullo, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

このプロトコルの結果に影響を与える最も重要な要因の一つは、出発原料として、新鮮な扁桃体の使用です。したがって、扁桃サンプルは手術後3時間以内に処理しなければなりません。扁桃腺は、大人と子供の両方から得ることができました。子供から扁桃物質が原因で扁桃腺（へんとう切除）の部分的な外科的除去に通常小さいです。そのため、多国籍企業の数が少ない扁桃摘出サンプル...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

<table><tbody><tr><td>Hanks balanced salt solution (HBSS)&amp;nbsp;</td><td>Gibco</td><td>14175-053</td><td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&amp;nbsp; &amp;nbsp;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td></tr><tr><td>Freezing medium</td><td></td><td></td><td>90% FBS and 10% DMSO</td></tr><tr><td>MACS buffer</td><td></td><td></td><td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td></tr><tr><td>PBSA&amp;nbsp;</td><td></td><td></td><td>PBS containing 0.2% BSA</td></tr><tr><td>PFA</td><td></td><td></td><td>PBS containing 1% paraformaldehyde (PFA). Make fresh. PFA is suspected carcinogen. Wear gloves and goggles.</td></tr><tr><td>Antibiotic-Antimycotic mix</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>60 mm Petridish</td><td>Nunc</td><td>150326</td><td></td></tr><tr><td>Dissecting foreceps</td><td>Fisher Scientific</td><td>1381241</td><td></td></tr><tr><td>Straight iris scissors&amp;nbsp;</td><td>Fisher Scientific</td><td>12912055</td><td></td></tr><tr><td>disposable scalpels</td><td>Swann-Morton</td><td>REF 0501</td><td></td></tr><tr><td>100 &amp;mu;m plastic cell strainer</td><td>Corning Life Sciences</td><td>352360</td><td></td></tr><tr><td>40 &amp;mu;m plastic cell strainers&amp;nbsp;</td><td>Corning Life Sciences</td><td>352340</td><td></td></tr><tr><td>2 ml plastic syringe</td><td>BD Biosciences&amp;nbsp;</td><td>300185</td><td></td></tr><tr><td>15 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62554502</td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>SARSTEDT</td><td>62547254</td><td></td></tr><tr><td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td><td>Thermo Scientific Heraeus Megafuge 16R</td><td></td><td></td></tr><tr><td>Ficoll&amp;ndash;Hypaque</td><td>Sigma-Aldrich</td><td>F5415-50ML</td><td>Ficoll solution</td></tr><tr><td>Fetal calf serum</td><td>Biological industries</td><td>040071A</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>SIGMA</td><td>D2650-5X5ML</td><td></td></tr><tr><td>MACS MS columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>MACS human CD3 MicroBeads</td><td>Miltenyi Biotec</td><td>130-092-881</td><td></td></tr><tr><td>MACS separator (Octo MACS)</td><td>Miltenyi Biotec</td><td>130-042-109</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Merck Millipore</td><td>1120180100</td><td></td></tr><tr><td>EDTA</td><td>AnalaR NORMAPUR</td><td>20302.293</td><td></td></tr><tr><td>CD2 conjugated to allophycocyanin (CD2-APC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>560642</td><td></td></tr><tr><td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>556632</td><td></td></tr><tr><td>Human serum&amp;nbsp;</td><td>Rockland Immunochemicals</td><td>D119-0100</td><td></td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F-530L</td><td></td></tr><tr><td>FACS tubes</td><td>BD Falcon</td><td>352003</td><td></td></tr><tr><td>Cryotube</td><td>SARSTEDT</td><td>72379</td><td></td></tr><tr><td>BD LSRII flowcytometer</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>BD FACSDiva 4.1 software</td><td>BD Biosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Nucleospin Blood&amp;nbsp;</td><td>Macherey-Nagel</td><td>740951.50</td><td>DNA isolation kit</td></tr><tr><td>TRIzol&amp;nbsp; reagent</td><td>Life technologies</td><td>15596</td><td>RNA isolation reagent</td></tr><tr><td>Hemocytometer</td><td>The Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>0610030</td><td>cell counter device</td></tr><tr><td>GelRed</td><td>Biotium</td><td>41003</td><td>Nucleic acid gel stain&amp;nbsp;</td></tr></tbody></table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

BおよびTリンパ球の高度に精製された亜集団における扁桃のMNC結果の効率的な分離。これは、（ 図3A）は、それぞれ、BおよびTリンパ球集団を検出するために、抗CD20および抗CD2抗体を用いたFACS分析によって確認しました。 BおよびTリンパ球の亜集団（ 図3B）の両方からの細胞の混合物である細胞画分中のMNCの結果とは対照的に、非効率的な分離。 <p class="jove_conten...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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15.7K ビュー.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ビデオ: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

体液性免疫のキープレーヤーの解明: 高度なマウス Peyer のパッチからのリンパ球の隔離のプロトコルを最適化

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

がん研究

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

トランスクリプトームプロファイリングのためのセザリー症候群患者からのヒト末梢血単核球およびCD4+ T細胞の単離

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

免疫・感染学

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

単離、分化、および血液からヒト抗体を分泌するB細胞の定量化：体液性免疫の機能読み出しなどのELISpot

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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.

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 ...

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

ヒト粘膜リンパ組織におけるEx Vivo自然免疫応答を研究する扁桃単核細胞の単核細胞の単離

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Isolation and Purification of B Cells from Human Peripheral Blood

Immediately after obtaining a 10-milliliter blood sample from the median cubital vein into a 15-milliliter tube containing potassium-supplemented EDTA, invert the tube several times to prevent clot formation. Then, add 35 milliliters of autoclaved red blood cell lysis buffer to the cells for a 5-minute incubation at room temperature.
When light can be observed through the tube, centrifuge the blood and confirm the presence of a white pellet. Remove the residual lysis buffer with 10 milliliters of autoclave to PBS, and resuspend the pellet in 10 milliliters of RPMI 1640 medium.
Plate the cells in a 10-centimeter culture dish for a 30-minute incubation at 37 degrees Celsius and 5% carbon dioxide. Then, gently swirl the culture dish a few times and transfer the floating cells into a plastic 15-milliliter conical tube.
After two washes by centrifugation, resuspend the pellet at a 5 to 10 x 106 cells per milliliter concentration in 200 microliters of cold PBS buffer, and add 5 microliters of biotinylated anti-human antibody cocktail specific for blood cells per 1 x 106 cells.
After a 30-minute incubation on ice, wash the cells in a 10-fold excess volume of sterile PBS, and resuspend the pellet in 5 microliters of streptavidin-conjugated microbeads per 1 x 106 cells for a 30-minute incubation on ice. At the end of the incubation, add 2 milliliters of PBS buffer to the cells and place the tube onto a magnetic stand for 8 minutes at room temperature.
When the microbeads have attached to the wall of the tube, carefully transfer the supernatant into a new conical tube, and add 2 more milliliters of PBS buffer to the cells. After the second supernatant collection, collect the pooled cells by centrifugation, and transfer the cells to a 5-milliliter polystyrene tube in fresh PBS buffer at a 1 x 107 cells per milliliter concentration.