DRWは、NIHの助成金AI089972とAI113217によってサポート、および粘膜免疫研究チームによると、バローズウェルカム基金からの医学者のためのキャリア賞を保持している。

注：ここで説明するすべての実験は、施設内動物管理使用委員会（IACUC）のガイドラインに従ったと動物研究小児病院（ARCH）、ボストン、マサチューセッツによって承認。 試薬の調製<ol><li> 1,000倍IL-4ストック：H 2 O、0.1％ウシ血清アルブミン（BSA）中に20μg/ mlにまで希釈IL-4サイトカイン。 -20℃で1.5 mlマイクロチューブやストアに200μlのアリコートで分注する。 </li><li> 1,000倍の抗CD40：4℃で1.0 mg / mlの、店でメーカーから入手します。 </li><li> B細胞刺激培地：ストレプトマイシンストック、5：RPMI-1640培地425mlの、新たに解凍したウシ胎児血清（FCS）を75mlを追加し、10ミリリットルの1.0 M HEPES緩衝液5mlのL-グルタミンストック5mlのペニシリンへ2-メルカプトエタノールの溶液最小必須培地非必須アミノ酸（MEM-NEAA）、及び3.5μlの。週間まで4℃でメディアを保管してください。 IL-4（20ng / ml）および抗CD40（1.0μg/ ml）を使用直前に加える唯一必要なメディアの容量に。 </li><li> 2％FCS-FACSバッファ：1X PBSの490ミリリットルにFCSの10ミリリットルを追加します。 4℃で保管してください。 </li><li> 2×トリプシン-EDTA溶液：1×PBS 20mlに5ミリリットル10xのトリプシンEDTAストック溶液（5.0グラムトリプシン、リットル当たり2.0グラムのEDTA）で希釈する。 4℃で一定分量の作業2倍に保管してトリプシン-EDTA。使用前に室温に温める。 </li><li>ウシ胎児血清：2週間まで4で解凍FCSしてください。細胞質の染色手順の前に氷の15分に置きます。 </li><li>中性緩衝ホルマリン：10％ホルマリン溶液（3.7％パラホルムアルデヒド）を準備します。ホルマリンは毒性があり、適切なPPEを装着したまま、したがって実験室ヒュームフードの下でそれを処理する。化学収納キャビネット内に室温で保存ホルマリン。 </li><li>メタノール：細胞は透過化される準備が整うまで-20℃で無水メタノール（100％）を保管してください。メタノールは可燃性で、適切な冷凍庫で保存する必要があることに注意してください。 </li></ol> 2。精製およびB細胞の活性化<ol><li>収穫したてのマウスの脾臓から、静かに3ミリリットルの注射器のプランジャーで70μmのセルストレーナーを通して臓器を押すことにより、単一細胞懸濁液を準備します。 15ミリリットルコニカル遠心管では、4℃で5分間300×gで1×PBSと遠心機の10ミリリットルで細胞を集める。 </li><li> 5分間の赤血球細胞溶解緩衝液1mlに上清と再懸濁ペレットをデカント。 5分間300×gで13.5 1X PBS mlの遠心で中和。 </li><li> （オプション）磁気（B220）抗CD45Rを用いB細胞を分離、製造業者の説明書に従ってMACS精製プロトコルに続いて、ビーズを標識。 </li><li> 37℃で5日間、IL-4（20ng / ml）および抗CD40（1.0μgの個/ ml）を含有する加温し、B細胞刺激培地中1.0×10 6細胞/ mlの精製されたB細胞を刺激する。 6ウェル培養プレートの2ウェル（各4ml）に分割し、培養を8mlで始まる。 </li><li> cultuチェック1.0×10 6細胞/ mlで維持するために毎日再。他のウェルに分割し、新鮮なIL-4および抗CD40を含有するB細胞刺激培地で培養を希釈して濃度を調整します。 </li></ol> 3.トリプシン処理、固定、および透過<ol><li>トリプシン処理<ol><li> 、15ミリリットルコニカルチューブで培養した細胞の1.0×10 7まで収集5分間300×gで遠心分離し、上清をデカント。 </li><li>細胞回収メディアから残留タンパク質を除去するために10mlのPBSで洗ってください。遠心分離工程を繰り返し、上清をデカント。 </li><li> （約100μl）をデカントした後の残存量の細胞ペレットを再懸濁します。 </li><li>慎重に細胞に室温800μlの0.1％（2倍）、トリプシンEDTAを追加します。円錐管の側を下に45°の角度でゆっくりと分注する。 </li><li>チューブを閉じ、液体がチューブ5-6時間の長さに沿って走るようにチューブを傾けることによって穏やかに混合S。糸、白色沈殿物が溶液中に形成することができる。分の下にトリプシン処理時間を制限する。 </li><li>冷たいFCSの1ミリリットルとのトリプシン処理反応を停止。冷PBSを10mlを加えることによって直ちにFCSを希釈する。 </li><li> 4℃で5分間300×gで遠心分離し、その後上清を除去し、氷にチューブを返す。 </li></ol></li><li>固定<ol><li>デカンテーション後の残留量の細胞を再懸濁。必要であれば、冷PBSで100μlにボリュームを起動します。 </li><li>ドラフト内で、細胞を10％中性緩衝ホルマリン溶液800μlを添加し、上下にピペッティング。チューブは、ホルマリンを添加すると暖めます。 10分間37℃でインキュベートする。 </li></ol></li><li>透過化<ol><li>緩やかに振盪速度でベンチトップボルテックスミキサーを設定します。 </li><li> 10ミリリットルの血清学的ピペットに冷たい（-20℃）メタノールの9ミリリットルを描画します。 </li><li>ボルテックスミキサーを介して細胞を含有するチューブを握り、ゆっくりとシャキーンを開始gの細胞。 </li><li>メタノールを連続的に混合されるように、管がまだ揺れている間に、それが細胞上に滴下して落下ようにチューブに冷メタノールを追加します。メタノールの最初の2ミリリットルの後に分注速度が遅いストリームに増加させることができる。 </li><li>チューブは透過処理を完了するために、30分間氷上で座ってみましょう。あるいは、細胞は、月まで-20℃で保存することができる。 </li></ol></li></ol> IgG1およびIgEのクラススイッチ4.蛍光染色<ol><li> 5分間300×gで遠心サンプルおよびメタノール溶液をデカント。白色沈殿物を細胞ペレットを得る。 </li><li>細胞を洗浄するために5分間300×gで10 PBS 1X室温mlの遠心分離器を追加します。一回繰り返します。注：室温PBSでペレットの洗浄は、ステップ4.1の後に形成することができる余分な沈殿物を溶解する。 </li><li> 2％FCS-FACS緩衝液10mlの追加の洗浄を行います。 </li><li> 96ウェル丸底プレートにサンプルを分配する5分間300×gで遠心する。上清を除去し、氷上にプレートを置く。 </li><li>以下の抗体希釈でウェルあたり2％FCS-FACS緩衝液60μlの中でステイン：抗IgM RPE / Cy7は、1：200、アンチIgG1のPE、1：600、抗IgE FITC、1：100、抗CD45R（B220）PerCPを-のCy5.5、1：200 </li><li>染色の5分後、300×gで5分間、2％FCS-FACSバッファーと遠心150μlでプレートを洗浄する。 </li><li> 5ミリリットルのFACSチューブに40μmのナイロン細胞ストレーナーを通して2％FCS-FACS緩衝液とピペット400μlの中で再懸濁します。 </li></ol> 5.次のゲーティング戦略を使用してください： <ol><li> FSC / SSCプロット（ 図1）上にあるブラストリンパ球集団からCD45R（B220）陽性細胞を分離します。 </li><li> B220陽性細胞からは、対応する集団を明らかにしたIgEおよびIgG1プロットを使用しています。 </li></ol>

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The authors have no disclosures.

抗CD40およびIL-4とともに培養中のマウス脾臓B細胞の刺激は、IgG1およびIgE 5へのクラススイッチを促進し、Tヘルパー2型（T H 2）の相互作用をシミュレートする。 B細胞は、総脾臓細胞12または精製された脾臓B細胞11の文脈においてCSRのために活性化することができる。プロトコル（段階2.3）で述べたように、B細胞の濃縮は、任意であり、それは有益だろうかど?...

マウスおよびヒトの免疫グロブリン重鎖（IgH遺伝子）クラススイッチ組換え（CSR）の間に、IgH遺伝子は 、定常領域エクソン（Cμ）が削除され、下流の定常領域エクソンのいくつかのセットの一つ（CのH秒 ）（ 例えば、Cγで置換されているμ他のIgクラス（ 例えば IgGを、IgEの、またはIgA）の産生に対するIgMの生産からスイッチになりCε、およびCα）、。 CSRは、C H 1のそれぞれの組の5 &#39;側に位置する1〜10キロバイト配列であるスイッチ（S）領域内で起こる。活性化誘導シチジンデアミナーゼ（AID）酵素は、シチジン脱アミノ化の活動を通じてCSRを開始します。 IgEはアレルギー疾患​​2の重要なメディエーターおよびIgEが産生され、アトピー性疾患への新しい治療的アプローチへの扉を開くことが規制されている方法の理解である。マウスとヒトでは、IgEが最も厳密に調節Igアイソタイプである。 IgEのは、通常、ティムのレベル数千で検出されるエス他のIgH未満は3アイソタイプが、高度に疾患状態4に上昇させることができる。しかし、IgEに対するCSRが完全には理解される。B細胞のインビトロ活性化を抗CD40またはLPSのいずれかと組み合わせてIL-4を使用して、IgG1およびIgEの5の両方にCSRを誘導する。活性化されたB細胞は、可溶性IgEがCSR後の培養中に分泌結合し、低親和性IgE受容体FcεRIIは/ CD23 2,6を 、表現する。フローサイトメトリーで分析する場合、したがって、受容体結合IgEとB細胞は、内因的なIgE 7を発現するB細胞にも同様に染色する。それは、マウスB細胞は、低親和性のIgG受容体FcγRIIB1（CD32）8を発現することが知られているが、我々の経験では、IgG1へのクラススイッチの測定を妨害しないようである。培養物中のB細胞活性化後のIgE切り替えを測定する場合しかし、非特異的表面結合IgE分析を不明瞭にすることができる。一般的な染色方法では、非IgEの発現細胞は、IgEのために積極的に染色する。 11 -ここで概説はCSRアッセイを実施し、マウス9から真のIgEを発現するB細胞を検出するために利用されている戦略である。トリプシンで活性化されたB細胞の治療は、一般的なラボの試薬は、タンパク質を消化するために、cytophylicおよび膜の両方に結合した表面IgEは削除されます。細胞質のIgEに対するその後の透過処理と染色は、このように真のIgE産生細胞を明らかにする。

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

Bリンパ球の免疫グロブリン重鎖（IgH遺伝子）クラススイッチ組換え（CSR）は、最初のIgMなどのIgA、IgE及びIgGのような他ののIgHアイソタイプ、に切り替わり表現方法である。 in vitroでのIgH CSRの測定は、分子および細胞免疫学の観点にDNA組換えおよび修復の範囲の生物学的プロセスの数を研究するための重要な方法である。 インビトロ CSRアッセイは、活性化B細胞のフローサイトメトリー測定表面Igの発現を含む。 IgAおよびIgGサブクラスの測定は簡単であるが、この方法によるIgEの測定はFcεRIIはへの結合により、可溶性IgEへの問題がある/ CD23は、活性化B細胞の表面に発現。ここでは、培養液中でCSRを受けたIgE産生マウスB細胞の正確な測定のためのユニークな手順を説明します。方法は、cyのによるIgE産生B系統細胞の検出を可能にする、細胞表面上のIgE-CD23複合体のトリプシン媒介開裂に基づくtoplasmic染色。この手順は、IgEに対するCSRのフローサイトメトリー分析のための便利なソリューションを提供しています。

この手順が正常マウスB細胞におけるIgEへのCSRを研究するために実施されている。先に説明したように11 CSRの測定効率を実証するために、我々は、抗CD40及びIL-4、マウス脾臓B細胞を刺激した。刺激の5日後、細胞を回収し、プロトコル蛍光標識されたIgM、IgG1を、IgEの、およびB220（CD45R）抗体を用いて上記の染色を用いて処理した。ブラストリンパ球（ 図1）にゲート、明確?...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.9K ビュー.  Brigham and Women's Hospital and Harvard Medical School. 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.

ビデオ: Detection of True IgE-expressing Mouse B Lineage 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

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

インフルエンザウイルスAおよびBの急速な分子の検出と分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

インフラマソーム活性化とマウス骨髄由来マクロファージにおける Pyroptotic の細胞死の検出

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

低コピーの検出数は B 型肝炎感染培養によって形成された統合ウイルス DNA

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

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, ...

Merkel Cell Polyomavirus Infection and Detection

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

メルケル細胞ポリオーマ ウイルス感染と検出

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...