私たちは親切tmuCD40 - リガンド- HeLa細胞細胞株を提供するために博士はクレメンスWendtnerに感謝。

脾細胞からマウスCD40活性化B細胞（mCD40B）を生成するためのプロトコルは2つの部分に分かれています：パート板として使用されるマウスCD40リガンド（CD40L）を発現するHeLa細胞（tmuCD40L HeLa細胞）の調製を示していますフィーダー細胞に結合した。パートBは、実際のマウスCD40 - Bの文化を説明します。 フィーダー細胞のA.準備（tmuCD40L HeLa細胞） tmuCD40L HeLa細胞は完全にコンフルエントになることはありません接着ヒト上皮細胞株、です。細胞は、そのため1週間に2回分かれています。以上の6週間以上の培養が推奨されていません。 <ol><li>滅菌ピペットを用いて初代培養から古い培地を除去し、1 × PBS 10 mLで細胞を洗浄。洗浄後PBSを吸引除去する。 </li><li> 37℃で10-15分では75 cm 2のフラスコに1 ×トリプシン/ EDTAの4 mLを加え℃に細胞を分離するために穏やかなタッピングを使用してください。 </li><li>野生型の培地10mLを追加し、トリプシンで消化を停止するように静かに回転さ。 </li><li>滅菌ピペットで50mLのチューブに細胞懸濁液を移し、7分間265 xgで細胞をスピンダウン。 </li><li>上清を除去し、野生型の培地10mlに細胞ペレットを再懸濁します。細胞懸濁液のアリコートの細胞数をカウントし、適切な数の細胞を3つの50 mLの試験管を準備します。 <ol><li>サブカルチャーのための2.5 × 10 6細胞</li><li>マウスCD40 - B細胞の培養に用いる/よく照射するための0.4 × 10 6細胞</li><li>フリーズして残り（必要に応じて）。 </li></ol></li><li> 7分間265 xgで細胞をスピンダウン。 </li><li>上清を取り除く。 <ol><li>継代のための：再懸濁し、2.5 × 10 6細胞を75センチメートル2細胞培養フラスコ（細胞密度2.5 × 10 5細胞/ mL）10 mLの選択培地で、37で細胞をインキュベート· CO 2 5％のCを。週2回のセルを分割する。 </li><li>マウスCD40 - B細胞の培養には：1つの6ウェルプレートに2.4 × 10 6個の細胞を必要とする。 0.2 × 10 6細胞/ mLの密度で野生型の媒体にtmuCD40L HeLa細胞を再懸濁し、78 Gyの時にそれらを照射する。よくそれぞれ、37でそれらをインキュベート° C、5％のCO 2に細胞懸濁液のプレートを2mL。 tmuCD40L HeLa細胞の接着性のある時にB細胞の刺激のために、この準備のプレートを使用する（少なくとも4時間：顕微鏡を用いて密着性を確認するには、B細胞の刺激を開始するために24時間以上待機しません）。 （bに進みます。） </li></ol></li></ol> B.マウスCD40 - Bの細胞培養 CD40刺激（0日）のための脾細胞のI.の準備： ご注意：あなたは、フィーダー細胞が接着していることを確かめる続行する前に。常にマウスインターロイキン-4、β-メルカプトエタノール、使用直前に増殖培地へのシクロスポリンの新鮮なソリューションを追加します。 <ol><li> C57BL / 6マウス（ハプロタイプH - 2b）の脾臓を摘出し、10〜20 mLのDMEMで二回を洗うには、別のチューブから10mLをピペットチップでそれを転送することにより、ゲンタマイシン[15μg/mL]で補足。マウスの汚染物質（例えば、髪の毛）を転送することは避けてください。セルストレーナー（100μmの）を通すことによって脾臓をミンチし、培地の別の10mLでストレーナーをすすぐ。室温で7分間、265 × gで脾細胞をスピンダウン、フィコール密度遠心分離によってmuCD40 - B細胞の培地、および別のリンパ球7mlのに再懸濁します。その30分を450 × gで15 mLの試験管や遠心機で5 mLのマウスPancoll（室温で予熱）上の層の細胞を室温でブレーキなしで行うには。 </li><li>界面で邪魔されずにリンパ球層を残し、上位層のほとんどをオフに描く。新鮮な50 mlチューブにリンパ球層を転送する、他の細胞を除去するために7分間265 × gでスピンダウンしてゲンタマイシン[15μg/mL]と30mLをDMEMで1回洗浄します。上清を捨て、mCD40 - B洗浄培地10mlに細胞を懸濁します。細胞懸濁液の一定量の細胞数を決定します。細胞懸濁液のアリコートをカウントすることにより、細胞数を決定します。 </li><li> 7分間265 xgで細胞の必要量をスピンダウン。 6ウェルプレート5の場合× 10 6細胞/ウェルこうしてプレート当たり30 × 10 6細胞、必要とされています。 </li><li>上清を除去し、新鮮防ぐために、成長因子、100μMのβ-メルカプトエタノールおよびサイクロスポリン0.63μg/ mLのようにインターロイキン-4の1 U / mLのを補充mCD40 - B培地で1.25 × 10 6細胞/ mLでリンパ球を懸濁します。 T -細胞の伸長（考えると濃度が1枚のMLの培地を参照してください！）。 </li><li> tmuCD40L HeLa細胞とのプレインキュベート6 - ウェルプレートから上清を取り除く。 </li><li>穏やかに6ウェルプレートの各ウェルにリンパ球懸濁液を4mL（1.25 × 10 6細胞/ mL）を追加します。 </li><li> 37プレートをインキュベート℃、5％のCO 2。 </li&gt; <li> 3日目に細胞は（II.1に進みます。）reculture。 </li></ol> II。 mCD40B細胞（3日目とその後週2回）のサブカルチャー： <ol><li>精力的に50 mlチューブに10 mLのピペットとプール一緒に上下にピペッティングにより収穫mCD40B細胞クラスター。 </li><li> 7分間265 xgでスピンダウンし、完全にmCD40 - B洗浄培地で上清を交換してください。アリコートの細胞の量をカウントしながら、7分間265 xgで細胞をスピンダウン。 0.75 × 10 6細胞/ mLの濃度でmCD40 - B培地で再懸濁しmCD40B細胞。 </li><li> 1 U / mLを、100μMのβ-メルカプトエタノール及び培地へのシクロスポリン0.63μg/ mLの濃度でマウスインターロイキン-4の新鮮なソリューションを追加。 </li><li> 6ウェルプレート照射tmuCD40L HeLa細胞でプレインキュベートから上清を取り除く。 </li><li>穏やかに6ウェルプレートの各ウェルにmCD40Bの細胞懸濁液を4mL（0.75 × 10 6細胞/ mL）を追加します。 </li><li> 37プレートをインキュベート℃、5％のCO 2。 </li><li>再び14日間の合計の後に高純度マウスCD40活性化B細胞で終わるために3〜4日ごとに細胞を継代培養。 </li></ol> C.トラブルシューティング：何かのCD40 - B細胞は増殖しないのですか？ <ol><li>あなたは、フィーダー細胞のCD40リガンド発現について確認しましたか？ </li><li> 24歳以上の刺激のために使用されるプレートに結合フィーダー細胞はありますか？ </li><li>マイコプラズマの混入はありますか？ </li><li>補充のために使用されるインターロイキン- 4ソリューションは、新鮮に解凍され、それは適切な生物活性を持っているのですか？ </li><li> β-メルカプトエタノールおよびシクロスポリン正しい濃度で添加されたのはいつですか？ </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="図1" /> 図1a。 <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="図1 B" /> 図1b。 <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="図1 D" /> 図1d。 <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="図2" /> 図2。

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実験は、実験動物の飼育のための国内及び欧州のガイドラインに従って行った。ドイツの動物福祉法を参照する動物は、適切な当局によって定期的に制御されていました。

このプロトコルの変更も、マウスCD40活性化B細胞の効率的な世代に降伏、特にCD40刺激し、使用されるマウス系統のタイプの起源について、可能な限りされています。アフマディら。 CD40 -リガンドマウスでトランスフェクションしたマウス線維芽細胞に対して同様の結果が示されている。このコンテキストでは外来の抗原の提示を確実に除外されますが、in vivoでのセキュリティと毒性?...

メディアのA.の準備： <table border="1"><tbody><tr><td> フィーダー細胞の野生型媒体 </td><td> フィーダー細胞を選択培地 </td><td></td></tr><tr><td> 試薬 </td><td> 濃度 </td><td> 試薬 </td><td> 濃度 </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L -グルタミン</td><td> 300μg/ mLの</td><td> L -グルタミン</td><td> 300μg/ mLの</td></tr><tr><td> FBS </td><td> 10パーセント</td><td> FBS </td><td> 10パーセント</td></tr><tr><td> HEPES </td><td> 10 mMの</td><td> HEPES </td><td> 10 mMの</td></tr><tr><td>ゲンタマイシン</td><td> 15μg/ mLの</td><td>ゲンタマイシン</td><td> 15μg/ mLの</td></tr><tr><td></td><td></td><td>ハイグロマイシンB </td><td>は0.2 mg / mLの</td></tr></table><table border="1"><tbody><tr><td> mCD40 - Bの洗浄媒体 </td><td> mCD40 - B培地 </td></tr><tr><td> 試薬 </td><td> 濃度 </td><td> 試薬 </td><td> 濃度 </td></tr><tr></tr><tr><td> D - MEM </td><td> </td><td> D - MEM </td><td> </td></tr><tr><td> L -グルタミン</td><td> 580μg/ mLの</td><td> L -グルタミン</td><td> 580μg/ mLの</td></tr><tr><td>グルコース</td><td> 4.5 mg / mLの</td><td>グルコース</td><td> 4.5 mg / mLの</td></tr><tr><td> HEPES </td><td> 10 mMの</td><td> HEPES </td><td> 10 mMの</td></tr><tr><td>ゲンタマイシン</td><td> 15μg/ mLの</td><td>ゲンタマイシン</td><td> 15μg/ mLの</td></tr><tr><td> </td><td> </td><td> MEM </td><td> 0.1mmの（1X） </td></tr><tr><td> </td><td> </td><td> FBS </td><td> 10パーセント</td></tr></tbody></table> B.その他の試薬： <table border="1"><tr><td> 試薬 </td><td> 会社 </td><td> オーダーNo </td></tr><tr><td> RPMI 1640 </td><td>インビトロジェンギブコ</td><td> REF 21875 </td></tr><tr><td> D - MEM </td><td>インビトロジェンギブコ</td><td> REF 41965 </td></tr><tr><td> Dulbeccos PBS（10倍） </td><td> PAA </td><td>猫なしH15 - 011 </td></tr><tr><td> Gencin </td><td>デルタの選択</td><td>アートなし7395800 </td></tr><tr><td> FBS </td><td>ロンザ</td><td>猫なしDE14 - 802C </td></tr><tr><td> HEPESバッファー</td><td> PAA </td><td>猫なしS11 - 001 </td></tr><tr><td> MEM NEAA </td><td>インビトロジェンギブコ</td><td> REF 11140 </td></tr><tr><td>ハイグロマイシンB </td><td> PAA </td><td>猫なしP02 - 015 </td></tr><tr><td>組換えマウスインターロイキン-4 </td><td> Immunotools </td><td>猫なし12340045 </td></tr><tr><td>シクロスポリン</td><td>ノバルティス</td><td>猫なしNDC 0078-0109-01 </td></tr><tr><td>トリプシン/ EDTA（10倍） </td><td>インビトロジェンギブコ</td><td> REF 15400-054 </td></tr></table> C.その他の用品： <table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> オーダーNo </td></tr><tr><td>滅菌ピペットチップ</td><td>ザルスタット</td><td></td></tr><tr><td> 6ウェルプレート</td><td> NUNC </td><td>猫なし140675 </td></tr><tr><td> 50mLのコニカルチューブ</td><td> BDファルコン™ </td><td>猫なし352070 </td></tr><tr><td>組織培養フラスコ</td><td>ザルスタット</td><td>猫なし83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

B細胞の研究は、CD40の活性化が、その抗原提示能を改善することが示されています。インターロイキン-4およびCD40リガンド（CD40L）で刺激したとき、ヒトB細胞は、高純度のCD40 - B細胞の非常に大きな金額に14日以内に末梢血の少量から困難なく拡張することができます（&gt; 10<sup&gt; 9</sup健康なドナーだけでなく、癌患者から患者あたり&gt;細胞）<sup&gt; 1月4日</sup&gt;。 CD40 - B細胞が重要なリンパ節ホーミング分子を発現し、T細胞を引き付けることができる<em&gt; in vitroで</em<sup&gt; 5</sup&gt;。さらに彼らは、効率的にT細胞に、プロセスと存在する抗原を取る<sup&gt; 6,7</sup&gt;。 CD40 - B細胞は素数ナイーブではないだけに示して、だけでなく、メモリーT細胞を展開していた<sup&gt; 8,9</sup&gt;。したがって、CD40活性化B細胞（CD40 - B細胞）の細胞ベースのimmunotherapy1、5,10のための免疫刺激抗原提示細胞（APC）の代替源として研究されている。 CD40 - B細胞はin vivoで効果的なT細胞応答を誘導し、我々はマウスのCD40活性化B細胞の産生のために細胞培養系を確立し、基礎となるメカニズムを研究するかどうかをさらに研究するために。脾細胞を用いてまたはCD40 -活性化のためのC57BL / 6マウス由来のB細胞を精製し、最適な条件は次のように同定された：C57BL / 6マウス（ハプロタイプH - 2b）はリンパ球の脾臓細胞からの起動は、密度勾配遠心法により精製し、HeLa細胞と共培養されています組換えマウスCD40リガンド（tmuCD40L HeLa細胞）を発現する細胞<sup&gt; 11</sup&gt;。細胞は3-4日ごとにreculturedされており、そのようなCD40L、インターロイキン-4のような主要なコンポーネントは、-メルカプトエタノールおよびサイクロスポリンが補充されています。このプロトコルでは、ヒトCD40 - B細胞（図1A、B）と同様のAPC -表現型と完全に活性化マウスCD40 - B細胞を（mCD40B）を取得する方法を示しています。 CD40刺激は、急速な伸長や細胞培養の14日以内に高純度（&gt; 90％）CD19 + B細胞（図1C、D）の拡大につながる。非トランスフェクト細胞の混入を避けるために、トランスフェクタントでマウスCD40リガンドの発現は、（図2）定期的に制御する必要があります。マウスCD40活性化B細胞はB細胞の活性化と分化と同様にin vitroおよびin vivoにおけるAPCとして機能する潜在能力を調査するための研究に使用することができます。また、彼らは腫瘍に対する治療剤又は予防接種を確立するための有望なツールを示しており、このアプローチの安全性と免疫原性に関する質問に答えるために役立ちます。<sup&gt; 12</sup&gt;。

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Murine Model of CD40-activation of B cells

このビデオでは、我々は、CD40 -活性化と免疫の誘導を研究するためにモデル抗原提示細胞（APC）として使用することができますC57BL / 6マウスの脾臓細胞からマウスB細胞、の拡張の手順を示しています。

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 ...

murine-model-of-cd40-activation-of-b-cells

Research

JoVE Journal

Immunology and Infection

12.6K ビュー.  University Hospital of Cologne. このビデオでは、我々は、CD40 -活性化と免疫の誘導を研究するためにモデル抗原提示細胞（APC）として使用することができますC57BL / 6マウスの脾臓細胞からマウスB細胞、の拡張の手順を示しています。

ビデオ: Murine Model of CD40-activation of B cells

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

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 immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

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 ...

免疫・感染学

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 ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

マウスノロウイルスのプラークアッセイ

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

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 ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

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, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

通常記憶 B 細胞の長命の形質細胞への分化モデル

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...