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...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 Culture Medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>Lipopolysaccharides from Escherichia coli</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>L6529</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>011-000</td>
<td>&nbsp;</td>
<tr>
<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553141</td>
<td>&nbsp;</td>
<tr>
<td>Rat Anti-Mouse IgG3</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553401</td>
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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 ...

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Immunology and Infection

18.0K 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

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

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

ヒト間葉系幹細胞（MSC）の免疫調節特性の評価

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外と体内の T, B, 骨髄細胞の抑制活性と移植から体液性応答の評価

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

生体外でマウス縁帯 B 細胞のせん断流動誘起移行の解析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.