我々は有用な議論のためのマーティンの研究室に感謝しています。この出版物は、保健研究のカナダ研究所（MOP - 89783）からの助成金によってサポートされています。 AMは、カナダのリサーチチェアティアII賞でサポートされています。

ステップI：磁気濃縮を経由して脾臓B細胞の分離を<ol><li>実験用マウスは、免疫システムの完全な成熟を確保するために8〜12週間熟成されるべきである。 </li><li>頚椎脱臼によりマウスを安楽死させると70％エタノールに浸漬。外科的腹部の左季肋部の皮膚と筋肉を通して切開を行うことによって脾臓を除去し、リン酸塩の3大部分にカット（PBS）緩衝生理食塩水。すべてのツールおよび試薬は無菌であることを確認してください。 </li><li> 1 mLのシリンジプランジャ、軽くマッシュPBSに70μmのセルストレーナーを介して脾臓のゴムの端を使用する。研削が大きい（増殖すなわち）細胞の破裂につながる可能性があるので押す動きではなく、研削モーションを使用してください。 </li><li> 5分間のこれ以上の350よりもgで細胞をスピンダウンし、PBSでペレットを再懸濁します。血球計数器を用いて再懸濁した細胞を数えます。 </li><li>キャップ付き滅菌5mLのポリスチレンチューブ（チューブあたり2 mLの最大音量）の5％正常ラット血清を含むPBSで1 × 10 8細胞/ mlの懸濁液を準備する。 </li><li>細胞のあらゆるmLのためのEasySep負の選択マウスのB細胞の濃縮カクテルの50μLを追加。混合物をピペット、チューブにキャップをし、15分間冷蔵庫に入れてください。 </li><li>細胞のあらゆる液を100μLEasySepビオチン選択カクテルを追加。混合物をピペット、チューブにキャップをし、15分間冷蔵庫に入れてください。 </li><li>細胞のあらゆるmLのEasySep磁性ナノ粒子を100μLを（ナノ粒子がよく混合され、溶液が均質であることを確認）を追加します。混合物をピペット、チューブにキャップをし、5分間冷蔵庫に入れてください。 </li><li> PBSで5分間EasySep磁石の位置と2.5mLのトップへソリューションを提供します。磁石とチューブを逆さにして新しいポリスチレンチューブに迅速に注ぐ。否定的に選択したセルが磁気チューブの壁にバインドされるようにチューブを振らないでください。 </li><li>さらに細胞懸濁液の純度を高めるために、管をさらに5分間磁石に再挿入し、新しいチューブに注いだことがあります。これは、全体の細胞の回復を減らすことができます。 </li><li> B細胞の純度は、B細胞表面マーカーのフローサイトメトリー分析によって評価することができる。濃縮に続いて、我々は一貫して&gt; 95％B220陽性細胞の純度を参照してください。 </li></ol> CFSE細胞の染色ステップII： <ol><li> mL当たり1 × 10 6細胞の最終濃度でアルブミン0.1％ウシ血清を添加した温かいPBSで分離した細胞を再懸濁します。 </li><li> CFSE（無菌ジメチルスルホキシドで希釈）の5mMのストック溶液を調製し、チューブ内の細胞のあらゆるmLの場合、2μlのを追加します。 10μMの最終濃度は、主要なB細胞の染色に最適です。 </li><li> 37℃暗所で10分間（注：CFSEは光に敏感であり、可能な場合は暗所に保管してください）​​。 </li><li>あなたの細胞に仔ウシの血清の等量を追加し、5分間氷上でインキュベートすることにより、汚れを癒やす。 </li><li> gを（レシピのステップIIIを参照）培養液のチューブを補充し、350で遠心分離で細胞を洗浄メディアの少なくとも5倍のボリュームに相当するが、仔ウシの血清の粘度を打ち消すようにし、細胞ペレットを生成するために追加しなければならないことに注意してください。 </li><li>培地で2回細胞を洗浄。セルは刺激のための準備が整いました。 </li></ol>ステップIII：LPSによる細胞の刺激を<ol><li> 10％ウシ胎児血清および50μMβ-メルカプトエタノール/を含むRPMI 1640を補完することによりin vitroで B細胞の培養液中で自分を準備します。 </li><li> 50μg/mLの最終濃度でLPSを追加して、刺激のメディアを準備します。フラットボトムの各ウェルを96ウェル組織培養プレートに刺激の培地のアリコートを125μL。 </li><li> 3.2 × 10 6細胞/ mlの最終濃度でLPSを含まない培地でCFSE染色B細胞を準備します。穏やかにピペッティングすることにより、刺激媒体とミックスを含むウェルに細胞懸濁液125μLを加える。 </li><li>各ウェルは、25μg/ mLの最終的なLPSの濃度で4 × 10 5個の細胞が含まれています。これらの値に変化が必要に応じて行うことができるが、これは、細胞増殖とクラスのスイッチングの最適なレベルを提供します。 </li><li> 37℃、72〜96時間、5％CO 2の細胞をインキュベート。 48時間後、大規模な増殖B細胞のクラスターは、顕微鏡下ではっきりと見えるようになります。 </li></ol>ステップIV：クラススイッチングのフローサイトメトリー分析<ol><li>あなたがクラススイッチを見て準備ができたら、彼らの文化の条件からあなたの細胞を除去し、染色バッファー（2％仔ウシ血清を含むPBS）1mLに二度、それらを洗ってください。 </li><li> 350グラムとインキュベーションでそれらをスピンダウンして、細胞、細胞をペレット化の非特異的な抗体染色を防ぐために5％正常マウス血清と氷上で15分間FcBlock per106細胞の1μgのでバッファを染色の100μLのTEそれら。細胞のブロッキングは氷上で行ってください。 </li><li>あなたの細胞を洗浄せずに、蛍光標識抗マウスIgG3抗体の10 6個の細胞あたり1μgのを追加し、細胞は氷上で30分間染色することができます。 </li><li>染色バッファーの200から300μLで遠心分離と再懸で細胞を2回洗浄する。セルはフローサイトメトリーによる評価のための準備が整いました。 </li><li> CFSEは最適波長492 nmで励起（アルゴンイオンレーザーによる）、517 nmで放出される。 IgG3抗体の励起および発光スペクトルは、その共役蛍光色素に依存しています。 </li></ol>代表的な結果磁気濃縮の後、細胞懸濁液は区別がつかない小さな円形の細胞（図1A）の均質な集団のようになります。刺激の48〜72時間後、拡大増殖細胞の分離されたクラスターは、（図1B）はっきりと見えるようになります。彼らはアポトーシスを起こすように、非増殖細胞の大部分は小さな粒度の細かい表示されます。 細胞のほとんどが7を死ぬことを開始する前に、CSRは、通常は刺激後72および96時間の間に最高レベルで検出することができます。この段階で、細胞は後に娘細胞の集団で現れる切り替え細胞と細胞分裂の数を受けているはずです。 CFSEで希釈し、LPS刺激の96時間後のIgG3の表面発現の代表的なフローサイトメトリープロットを図2に見ることができます。 表1。カクテルを切り替えアイソタイプ。注：覚せい濃度値は、推奨事項のみです。滴定が必要になる場合があります。 <table border="1"><tbody><tr><td>希望のアイソタイプ</td><td>刺激のカクテル</td></tr><tr><td> IgG1and IgE値</td><td> LPS（25μg/mL）およびIL - 4（10 ng / mL）を</td></tr><tr><td> IgG1and IgE値</td><td>抗CD40（10μg/mL）およびIL - 4（10 ng / mL）を</td></tr><tr><td>的なIgG2a </td><td> LPS（25μg/mL）およびIFN -γ（10 ng / mL）を</td></tr><tr><td> IgG2b及びIgG3に</td><td> LPS（25μg/mL） </td></tr><tr><td> IgAの</td><td> LPS（25μg/mL）、TGF -β（2 ng / ml）およびIL - 5（1.5 ng / mL）を</td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="図1" /> 図1。単離及び脾臓B細胞のLPS刺激。 （）磁気LPSの刺激の前に脾臓B細胞を濃縮。 （B）48時間LPSの25μg/ mLので刺激した後。増殖する細胞は、ブラストおよびアポトーシス細胞のバックグラウンドでのクラスターとして見られている。 <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="図2" /> 図2。96時間後の増殖とクラススイッチ組み換え。 （A）LPSのためのCFSEの希釈は、文化の中で96時間後に細胞を刺激した。それぞれ独立したピークは、独立した細胞分裂に対応するCFSEの蛍光の希釈を表しています。 （B）細胞分裂のいくつかのラウンドに続くIgG3に肯定的な人口の出現を示すIgG3に表現。

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, 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=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., 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=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

利害の衝突は宣言されません。

上記で概説プロトコルは、プライマリマウスB細胞のクラススイッチの間にAIDの発現と機能を解析する標準的なアッセイを提供する。刺激のメディアへの変更は、他のアイソタイプ（表1 8、9に示す）に切り替える誘導できるものはありますが、このプロトコルでは、IgMからIgG3にするためにスイッチング誘導する細菌のLPSを使用しています。 我々がいることを指摘...

<table><tbody><tr><td>Phosphate Buffered Saline</td><td>GIBCO, by Life Technologies</td><td>14190</td><td></td></tr><tr><td>EasySep Mouse B Cell Enrichment Kit</td><td>Stem Cell Technologies</td><td>19754</td><td></td></tr><tr><td>Polystyrene tubes</td><td>BD Biosciences</td><td>352058</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>CellTrace CFSE Cell Proliferation Kit</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>Bovine Calf Serum</td><td>Hyclone</td><td>SH30072.03</td><td></td></tr><tr><td>RPMI 1640 Culture Medium</td><td>GIBCO, by Life Technologies</td><td>11875</td><td></td></tr><tr><td>Lipopolysaccharides from &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>L6529</td><td></td></tr><tr><td>&amp;#946;-mercapt&amp;#8211;thanol</td><td>GIBCO, by Life Technologies</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td></td></tr><tr><td>Normal Mouse Serum</td><td>Jackson ImmunoResearch</td><td>011-000</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Rat Anti-Mouse IgG3</td><td>BD Biosciences</td><td>553401</td><td></td></tr></tbody></table>

induction and assessment of class switch recombination in purified murine b cells

体液性免疫はB細胞によって維持されると抗体の分泌を介して免疫系の分岐です。 B細胞の活性化により、免疫グロブリンの遺伝子座は、結合能と分泌される抗体のエフェクター機能を変更するために遺伝的改変のシリーズを受ける。このプロセスは、デフォルトのIgM抗体のアイソタイプがIgG抗体は、IgA、またはIgEのいずれかに置換されているクラススイッチ組み換え（CSR）として知られているゲノムの組換え事象によって強調表示されます。それぞれのアイソタイプは、それによって免疫の維持にCSRが重要な意思異なるエフェクター機能を持っています。 免疫グロブリン遺伝子座の多様化は、酵素活性化誘導シチジンデアミナーゼ（AID）によって媒介される。詳細にこのプロセスを説明する回路図ビデオがオンラインで入手可能（http://video.med.utoronto.ca/videoprojects/immunology/aam.html）です。 AIDの活動とCSRの経路は、一般的にB細胞の機能とマウスにおける液性免疫の評価で検討されています。このレポートに記載されているプロトコルは、IgG3（他の抗体アイソタイプについては表1を参照）に切り替えるクラスを誘導する細菌のリポ多糖（LPS）をマウス脾臓とその後の刺激からのB細胞の単離の方法を提示。さらに、蛍光細胞染色色素カルボキシフルオレセインスクシンイミジルエステル（CFSE）が刺激された細胞の細胞分裂、スイッチング1、2をアイソタイプに重要なプロセスを監視するために使用されます。 AIDとCSRが発生するメカニズムの規制はまだ明らかではないため、in vitroでのクラススイッチのアッセイで 、様々なマウスモデルでこれらのプロセスをテストするための信頼性の高い方法を提供する。これらのアッセイは、以前は次のノックアウトマウス3を用いて遺伝子の欠損のコンテキストで使用されている。さらに、B細胞のスイッチングのin vitroでのRNAノックダウンまたは導入遺伝子の発現4-6に遺伝子発現を調節するためにウイルスの伝達を付けることができます。実験のこれらのタイプからのデータは、AIDの活動、CSRの反応の解像度、およびマウスにおける抗体媒介性免疫の理解に影響を及ぼしている。

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

抗原曝露後、活性化B細胞の亜集団は、異なるエフェクター機能を有する抗体のアイソタイプを生成するためにクラススイッチ組み換え（CSR）として知られているプロセスを経る。このレポートに記載されているプロトコルは、CSRを誘導して分析できる方法について説明します。<em&gt; in vitroで</em&gt; B細胞機能を研究の目的のために。

マウスB細胞を精製における誘導とクラススイッチ組み換えの評価

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

induction-assessment-class-switch-recombination-purified-murine-b

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.1K ビュー.  University of Toronto. 抗原曝露後、活性化B細胞の亜集団は、異なるエフェクター機能を有する抗体のアイソタイプを生成するためにクラススイッチ組み換え（CSR）として知られているプロセスを経る。このレポートに記載されているプロトコルは、CSRを誘導して分析できる方法について説明します。 in vitroで B細胞機能を研究の目的のために。

ビデオ: Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

B細胞の組換えレトロウイルスの生産と感染症

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

免疫・感染学

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

特定の遺伝子の過剰発現やノックダウン後にラモスB細胞に体細胞高頻度変異の評価

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

真のIgE発現するマウスB系統細胞の検出

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

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 ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

マイクロ Rna と受ける DNA の組換えをクラス スイッチしてプラズマ細胞の分化誘導された B 細胞における Mrna のゲノム解析の HDAC 阻害剤を介した変調

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6マウスにおける全身性エリテマトーデス（SLE）のBM12誘導モデル

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

医学

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリン アイソタイプ反応の評価

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

マウスB-1a細胞におけるCXCR4のレトロウイルス過剰発現と骨髄およびIgM産生への標的B-1a細胞の移行のための養子転移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

マウスペイヤーパッチからの胚芽センターB細胞のJH4イントロンにおける体細胞ハイパー突然変異の解析

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

マウスB細胞を精製における誘導とクラススイッチ組み換えの評価

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この動画の章

B細胞の組換えレトロウイルスの生産と感染症

特定の遺伝子の過剰発現やノックダウン後にラモスB細胞に体細胞高頻度変異の評価

真のIgE発現するマウスB系統細胞の検出

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

単離とマウスリンパ球の活性化

マイクロ Rna と受ける DNA の組換えをクラススイッチしてプラズマ細胞の分化誘導された B 細胞における Mrna のゲノム解析の HDAC 阻害剤を介した変調

C57BL / 6マウスにおける全身性エリテマトーデス（SLE）のBM12誘導モデル

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリンアイソタイプ反応の評価

マウスB-1a細胞におけるCXCR4のレトロウイルス過剰発現と骨髄およびIgM産生への標的B-1a細胞の移行のための養子転移

マウスペイヤーパッチからの胚芽センターB細胞のJH4イントロンにおける体細胞ハイパー突然変異の解析

マウスB細胞を精製における誘導とクラススイッチ組み換えの評価

要約

さらに動画を探す

この動画の章

B細胞の組換えレトロウイルスの生産と感染症

特定の遺伝子の過剰発現やノックダウン後にラモスB細胞に体細胞高頻度変異の評価

真のIgE発現するマウスB系統細胞の検出

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

単離とマウスリンパ球の活性化

マイクロ Rna と受ける DNA の組換えをクラス スイッチしてプラズマ細胞の分化誘導された B 細胞における Mrna のゲノム解析の HDAC 阻害剤を介した変調

C57BL / 6マウスにおける全身性エリテマトーデス（SLE）のBM12誘導モデル

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリン アイソタイプ反応の評価

マウスB-1a細胞におけるCXCR4のレトロウイルス過剰発現と骨髄およびIgM産生への標的B-1a細胞の移行のための養子転移

マウスペイヤーパッチからの胚芽センターB細胞のJH4イントロンにおける体細胞ハイパー突然変異の解析

マイクロ Rna と受ける DNA の組換えをクラススイッチしてプラズマ細胞の分化誘導された B 細胞における Mrna のゲノム解析の HDAC 阻害剤を介した変調

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリンアイソタイプ反応の評価