この作品は、DNAフィンガープリントおよび診断のためのバイオテクノロジー本部、インド政府とセンターのコア·ファンドからの革新的な若手生物工学研究者賞BT/BI/12/040/2005とBT/PR13289/BRB/10/745/2009助成金によって支えられて、ハイデラバード。 MNR GBはマニパル大学の博士号の学位を追求に向けた科学産業研究評議会のジュニアとシニアリサーチフェローの受信者です。 SBはマニパル大学の博士号の学位を追求に向けたバイオテクノロジー本部のジュニアとシニアリサーチフェローシップ受賞。

これは、Cを実行することをお勧めしますバイオセーフティ封じ込めレベル2（BSL-2）との実験室でグラブラタ感染実験。 <ol><li> THP-1マクロファージ単層の調製。 </li><li> Cの準備グラブラタ細胞懸濁液。 </li><li> CとTHP-1マクロファージの感染グラブラタ細胞。 </li><li>コロニー形成単位アッセイによって貪食率および細胞内複製を測定する。 </li><li>共焦点レーザー走査顕微鏡を用いて細胞内複製のモニタリング。 </li></ol> 1。 THP-1マクロファージ単層の調製<ol><li> THP-1は急性単球性白血病を患っている14 1歳の男児の末梢血由来のヒト単球細胞株である。 THP-1懸濁細胞株は、日常的に、10％FBS（ウシ胎児血清）を補充したRPMI-1640培養培地中、加湿5％CO 2中37℃で維持する。ホルボール12 - myristaによる治療テ13 - アセテート（PMA、ホルボールエステル）がマクロファージにTHP-1単球細胞の最終分化をもたらす。重要なことに、PMA処理は細胞生存率に影響を与えず、分化した細胞は分裂しない。 </li><li>シードRPMI-1640完全培地中で100mmの培養皿中約1.5×10 6個のTHP-1単球（10％血清、2mMグルタミンおよび1×抗生物質を補充したRPMI-1640培地、10ミリリットル/ディッシュ）細胞を、37℃で増殖およびlet 2日間、5％CO 2でCが°。 </li><li> THP-1細胞を8×10 5細胞/ mlの密度に達した後、細胞培養層流フードに培養皿に移し、穏やかに沈降した細胞を再懸濁するためにそれを旋回させる。 </li><li>細胞懸濁液をピペットで、4分間1,000 rpmで（130×g）で15ミリリットル滅菌チューブと遠心分離機に入れる。上澄み液を捨て、5ミリリットルの新鮮な、予め温め、RPMI-1640完全培地中のTHP-1細胞ペレットを一時停止。 </li><li>血球計数器を用いて細胞を列挙し、セル画を希釈予め加温したRPMI-1640完全培地で10 6細胞/ mlの最終濃度までリットルの懸濁液</li><li> （ホル12 - ミリステート13 - アセテート溶液は、ジメチルスルホキシドで製造）10ミリリットルのTHP-1細胞懸濁液（最終濃度16 nM）を160 mMのPMAストックを1μLを加え、よく混ぜる。 24ウェル組織培養プレートの各ウェルに細胞懸濁液1mlを分配し、12時間、5％CO 2、37℃で維持したインキュベーター中でプレートをインキュベートする。 </li><li>古いメディアを削除するには、1ミリリットルのRPMI-1640完全培地を予め暖め追加し、細胞を5％CO 2中37℃で12時間かけて回復することができます。 </li><li>懸濁液中のその楕円形のTHP-1単球細胞は平らに、紡錘型、付着マクロファージに分化したことを確認するために倒立顕微鏡下で細胞を観察します。これらの分化した細胞は、現在で調査を行って準備ができているグラブラタ感染実験。 </li></ol> 2。 Cの準備グラブラタ ＆＃160;細胞懸濁液 C.グラブラタ野生型株のBG2は、THP-1マクロファージを感染させるために使用される。C.グラブラタ細胞は、液体YPD（酵母エキス（1％）、ペプトン（2％）、デキストロース（2％））培地中で日常的に培養される。固形YPD培地は、オートクレーブ処理前に培地、2％寒天を添加することにより調製される。 <ol><li> Cの文化を準備するにはグラブラタ 、滅菌された使い捨ての耳で寒天プレートからシングルコロニーを上にピックアップし、10ミリリットルにYPD液体培地に接種し、30℃で（200回転）しながら14〜16時間インキュベート</li><li>テーブルトップマイクロ遠心で5分間4,000 rpmで（1,800×g）で遠心この培養物（1ml）を、滅菌PBS（リン酸緩衝食塩水、pH 7.4）で3回細胞を洗浄し、1mlのPBS中に細胞ペレットを再懸濁する。 </li><li> C.のOD 600を測定しますグラブラタ細胞懸濁液を2×10 6細胞/ mlの密度を得るために、滅菌PBSの適切な容量で希釈した（OD 600= 0.1）。あるいは、所望の細胞密度を、血球計数器を用いて酵母細胞を計数することによって達成することができる。 </li></ol> 3。 CとTHP-1マクロファージの感染グラブラタ細胞<ol><li> 50μLCを追加0.1のMOI（感染多重度）を取得し、5％CO 2、37℃でプレートをインキュベートするTHP-1マクロファージ（24ウェル培養プレートのウェルに播種10 6細胞）にグラブラタ細胞懸濁液。 </li><li>実行可能な酵母数を決定するために、50μlの温度を希釈グラブラタ細胞懸濁液を滅菌PBS中で100倍にYPD固体培地上に100μlをプレート。 30℃で培養すると、手動で1〜2日後に現れる酵母コロニーの数を数える。これらの数は、今後、0時間のC.として呼ばれるグラブラタのCFU（コロニー形成単位）。 </li><li> CとTHP-1細胞の2時間の共培養の後24ウェル組織培養プレートを反転させることにより細胞グラブラタ 、廃棄培地iを貯水池Naおよびnonphagocytosed細胞外Cを削除するに予熱無菌PBSで3回優しく洗うグラブラタ細胞。 PBSで穏やかな洗浄は、THP-1マクロファージ単層に損傷を引き起こすことなく、全ての細胞外酵母の完全な除去を達成するために必要不可欠である。 </li></ol> 4。コロニー形成単位アッセイを経由して貪食率と細胞内複製の測定<ol><li>溶解温度 1ml中グラブラタ感染THP-1マクロファージ2分間の滅菌H 2 O、ならびにマクロファージから破片を除去し、微量遠心管中で細胞溶解物を収集するために穏やかにこすり。完全なマクロファージ溶解の微視的な検証は、すべての内部化酵母の回復のために極めて重要である。 </li><li>滅菌PBS中の溶解液の100倍希釈液を調製、YPD固体培地上に100μlをプレートに30℃で培養する</li><li> 1-2日後、手動C.数を数えるそのAP グラブラタコロニーYPD培地にしまっ、希釈倍率（2時間のCFU）で乗算し、Cのパーセントを指す貪食率を計算する以下の式を用いて2時間の同時インキュベーションの後にTHP-1マクロファージによって摂取されるグラブラタ細胞。 ％の貪食= [（2時間でのCFU）/（0時間でのCFU）]×100 </li><li> C.の細胞内複製の速度を測定するためにTHP-1マクロファージにおけるグラブラタ細胞が、ポイントは、感染、すなわちポスト異なる時間に、細胞内の酵母を収集します。 4、6、8、10、12、および24時間、3.3から4.3の手順を繰り返すこと。 </li><li> Cの倍生存/複製を計算する2時間（貪食酵母）におけるそれらと任意の所与の時点での合計のCFUを分割することによりTHP-1マクロファージにおける細胞グラブラタ 。 </li></ol> 5。共焦点ラスを使用して、細胞内複製を監視するER走査顕微鏡<ol><li>第1節に記載の手順に従って、2つの4ウェルチャンバースライドの各ウェル内のPMA処理した5×10 5 THP-1細胞を播種し、12時間、5％CO 2、37℃でインキュベートする。 </li><li>あらかじめ温めておいたRPMI-1640完全培地で古い培地を交換し、細胞は12時間、PMA処理から回復することができます。 </li><li>調製GFP（緑色蛍光タンパク質）タグ付きC.グラブラタ細胞懸濁液2章で説明され、1のMOIのTHP-1マクロファージに感染するように。 C.もしGFPを発現するグラブラタ株が利用できない、FITC（フルオレセインイソチオシアネート）で標識された酵母細胞は、感染を転記すなわち初期の事象を監視するために使用することができる。 2時間で貪食率およびファゴリソソーム成熟。 </li><li> 5％CO 2、37℃で2時間スライドをインキュベート培地を廃棄し、滅菌で3回洗浄するために注意深く反転し、PBSで予め温め。 </li><li> RPMI-1640完全MEDIを予め温め500μLを追加UM 1スライドの各チャンバーにし、22時間、5％CO 2、37℃で静置する。 </li><li> 2時間Cを修正するにはグラブラタ感染THP-1マクロファージは、他のスライドの各チャンバーに（PBS中で調製）3.7％ホルムアルデヒドを500μlを加え、暗所で20分間室温で静置する。 </li><li>洗浄は、PBSで3回スライドさせ、500μlのトリトン-X（0.7％）を添加し、暗所で5分間室温でインキュベートする。 </li><li> PBSで洗浄スライド3X、暗闇の中で3〜5分間乾かし培養スライドや空気から容器を外します。 </li><li>注意深くスライド回避気泡形成にDAPI（4 &#39;、6 - ジアミジノ-2 - フェニルインドール）を含むベクタシールド封入剤を用いてカバースリップをマウント。優しくキムワイプで過剰な液体を除去し、マニキュアでカバースリップの端をシールします。使用するまで4℃の暗所に保管してスライド。 </li><li>繰り返しますTHP-1細胞24時間後に感染を処理するために5.4および5.6から5.9を繰り返します。 </li><li>レーザ走査confoを用いて画像セルCAL顕微鏡（60X油浸対物レンズ、DAPI及びGFP染色のための405 nmおよび488 nmで励起、それぞれ）。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

自然免疫系は、日和見真菌感染症の制御に重要な役割を果たしている。マクロファージは、摂取および真菌病原体の破壊によって防衛を抗真菌に貢献。このように、生存および/またはマクロファージの抗菌機能に対抗するために必要な要因の解明は、真菌の病原性戦略の我々の理解を進める。この文脈において、我々は、ヒトの日和見真菌病原C.の相互作用を特徴づけるために、ヒト?...

カンジダ種は、免疫不全患者の生命を脅かす侵襲性真菌感染症の1の主要な原因である。 カンジダ·グラブラタ 、新興院内病原体は、地理的な位置1-3に応じて、集中治療室の患者からの第二または第三の最も頻繁に孤立したカンジダ種である。系統発生的に、C.グラブラタ 、半数体出芽酵母、より密接に病原性カンジダ属に比べcerevisiaeの非病原性のモデル酵母サッカロミセスに関連しています。Cを含むカンス 4。これと一致して、C.グラブラタ交配 、分泌されたタンパク質分解活性及び形態学的可塑性4-5を含むいくつかの重要な真菌の病原性の特徴を欠いている。 C.もののグラブラタが菌糸を形成しない、それが生き残るためには、6〜8は、それが独特の病因メカニズムを開発したことを示唆しているマウスおよびヒトのマクロファージ内で複製することができます。限られた情報は、C.戦略について提供されていますグラブラタは、栄養の乏しい細胞内マクロファージ環境を生き残るためには、酸化および5宿主免疫細胞によって取り付けられた非酸化宿主応答に対抗するために採用しています。関連マクロファージモデル系は、Cの相互作用を描写する前提条件である機能的ゲノムおよびプロテオミクスのアプローチを介してホスト貪食細胞とグラブラタ 。末梢血単核細胞（PBMC）及びヒトおよびマウス由来の骨髄由来マクロファージ（BMDMs）は、それぞれ、以前のC.の相互作用を研究するために使用されている宿主免疫細胞7,9とグラブラタ 。しかし、PBMCおよびBMDMsを得ることが困難、その限られた寿命と異なる哺乳動物ドナー間本質的な変化は、これらの細胞の活用など多彩なモデルシステムを制限します。 ここでは、intracellulを研究するためのin vitro系の確立のための方法を説明しますCの ARの挙動ヒト単球細胞株THP-1由来マクロファージにおける細胞グラブラタ 。このプロトコルの全体的な目標は、簡単にホスト真菌病原体の相互作用の様々な側面を研究するために操作することができる、簡単で、安価、迅速、かつ再現性の細胞培養モデル系を制定することでした。 THP-1細胞は、以前は、細菌、ウイルス、および真菌10-12を含む広範囲の病原体に対する宿主免疫応答を解読するために使用されている。単球THP-1細胞を維持するのに容易であり、ヒトの単球由来のマクロファージを模倣し、適切なマクロファージマーカー13を発現するマクロファージ、ホルボールエステル治療の際に、区別することができる。 THP-1マクロファージモデル系の主な利点は、使いやすさと洗練された機器の必要がないことである。 ここで紹介するプロトコルは、HOSで、他の人間の真菌病原体の相互作用を研究する容易に適応される免疫細胞をトン。現在の手順はまた、ハイスループット変異スクリーニングを使用して、関心対象の病原体の病原性因子を同定するために用いることができる。この概念実証は、ヒトマクロファージ8にC.グラブラタの生存のために必要とされる56遺伝子のセットを同定するために、THP-1培養モデルシステムの使用の成功により例示した。

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="39" style="height:40px;width:163px;">THP-1</td>
			<td style="width:105px;">American Type Culture Collection</td>
			<td style="width:85px;">TIB 202</td>
			<td style="width:243px;">Human acute monocytic leukemia cell line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">RPMI-1640</td>
			<td style="width:105px;">Hyclone</td>
			<td style="width:85px;">SH30096.01</td>
			<td style="width:243px;">For maintaining THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Phorbol 12-myristate 13-acetate</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">P 8139</td>
			<td style="width:243px;">Caution: Hazardous</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">YPD&#160;</td>
			<td style="width:105px;">BD-Difco</td>
			<td style="width:85px;">242710</td>
			<td style="width:243px;">For growing Candida glabrata cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Formaldehyde</td>
			<td style="width:105px;">Sigma-Aldrich</td>
			<td style="width:85px;">F8775</td>
			<td style="width:243px;">For fixation of C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Phosphate buffered saline (PBS)</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,&#160; pH 7.4) for washes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Saline-sodium citrate (SSC)&#160;</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Prehybridization buffer</td>
			<td style="width:105px;"></td>
			<td style="width:85px;"></td>
			<td style="width:243px;">Buffer (50% formamide, 5x Denhardt&#8217;s solution, 5x SSC, 1% SDS) for hybridization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">VECTASHIELD mounting medium</td>
			<td style="width:105px;">Vector Labs</td>
			<td style="width:85px;">H-1200</td>
			<td style="width:243px;">For mounting slides for confocal microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">32P-labeled &#945;-dCTP</td>
			<td style="width:105px;">JONAKI-BARC</td>
			<td style="width:85px;">LCP-102</td>
			<td style="width:243px;">For radiolabeling of signature tags</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">100 mm tissue culture dishes</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">430167</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">24-well tissue culture plate</td>
			<td style="width:105px;">Corning</td>
			<td style="width:85px;">3527</td>
			<td style="width:243px;">To perform C. glabrata infection studies in THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">4-chamber tissue culture-treated glass slide</td>
			<td style="width:105px;">BD Falcon</td>
			<td style="width:85px;">REF354104</td>
			<td style="width:243px;">To image C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hemocytometer</td>
			<td style="width:105px;">Rohem India</td>
			<td style="width:85px;"></td>
			<td style="width:243px;">For enumeration of cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Table top microcentrifuge</td>
			<td style="width:105px;">Beckman Coulter</td>
			<td style="width:85px;">Microfuge 18</td>
			<td style="width:243px;">For spinning down cells in microtubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Table top centrifuge</td>
			<td style="width:105px;">Remi</td>
			<td style="width:85px;">R-8C</td>
			<td style="width:243px;">For spinning down cells in 15 ml tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Spectrophotometer</td>
			<td style="width:105px;">Amersham Biosciences</td>
			<td style="width:85px;">Ultraspec 10</td>
			<td style="width:243px;">To monitor absorbance of yeast cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Plate incubator</td>
			<td style="width:105px;">Labtech</td>
			<td style="width:85px;">Refrigerated</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Shaker incubator</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:85px;">Innova 43</td>
			<td style="width:243px;">To grow C. glabrata cells</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Water jacketed CO2&#160; incubator</td>
			<td style="width:105px;">Thermo Electron Corporation</td>
			<td style="width:85px;">Forma series 2</td>
			<td style="width:243px;">To culture THP-1 cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Confocal microscope</td>
			<td style="width:105px;">Carl Ziess</td>
			<td style="width:85px;">Ziess LSM 510 meta&#160;</td>
			<td style="width:243px;">To observe C. glabrata-infected THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Compound microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:85px;">CKX 41</td>
			<td style="width:243px;">To observe C. glabrata and THP-1 macrophages</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">PCR machine</td>
			<td style="width:105px;">BioRad&#160;</td>
			<td style="width:85px;">DNA Engine</td>
			<td style="width:243px;">To amplify unique tags from input and output genomic DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">Hybridization oven</td>
			<td style="width:105px;">Labnet</td>
			<td style="width:85px;">Problot 12S</td>
			<td style="width:243px;">For hybridization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:163px;">PhosphorImager</td>
			<td style="width:105px;">Fujifilm</td>
			<td style="width:85px;">FLA-9000</td>
			<td style="width:243px;">For scanning hybridized membranes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Thermomixer</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:85px;">Thermomixer Comfort</td>
			<td style="width:243px;">For denaturation of radiolabeled signature tags</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:163px;">Gel documentation unit</td>
			<td style="width:105px;">Alphainnontech</td>
			<td style="width:85px;">Alphaimager</td>
			<td style="width:243px;">To visualize ethidium bromide-stained DNA in agarose gels</td>
		</tr>
	</tbody>
</table>

establishment of an in vitro system to study intracellular behavior of candida glabrata in human thp-1 macrophages

細胞培養モデル系では、ホスト環境条件の近隣模倣場合、微生物病原体感染の特定の段階の研究のための動物モデル系を、安価で再現性があり、容易に操作可能な代替物として機能することができる。 、ホルボールエステル治療の際に、マクロファージに分化されたTHP-1ヒト単球細胞株は、以前に結核菌を含む多くの細胞内病原体の病原性戦略を研究するために使用されている。ここでは、in vitro細胞培養モデルを制定するためのプロトコルを議論ホスト貪食細胞と日和見人間酵母病原体のカンジダ·グラブラタの相互作用を記述するために、THP-1マクロファージを用いたシステム。このモデル系は、簡単、迅速、ハイスループット変異スクリーニングに適しており、まだ高度な機器を必要としない。典型的なTHP-1マクロファージ感染実験を回収し、細胞内を可能にするために、追加の24〜48時間で約24時間を要するコロニー形成単位ベースの実行可能性の分析のための富栄養培地上で増殖する酵母。他のin vitroモデル系と同様に、このアプローチの可能な限界は、ヒト宿主に存在する非常に複雑な免疫細胞回路に得られた結果を外挿することが困難なことである。しかし、これにもかかわらず、現在のプロトコルは、真菌病原体が、抗菌応答に対抗して生き残る/回避適応し、宿主免疫細胞の貧栄養環境下で増殖するように使用することができる戦略を解明することは非常に便利です。

CとPMA処理したTHP-1マクロファージの感染分析グラブラタ野生型（wt）細胞は、wt細胞を、2時間の同時インキュベーション後に55〜65％の割合でマクロファージによって貪食されたことを明らかにした。さらに、C.グラブラタ細胞は、THP-1マクロファージによる殺傷に抵抗することができましたし、中程度の5を受けた- THP-1マクロファージ8と共培養の24時?...

Watch this Scientific Journal Video about Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages at JoVE.com

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

現在の記事は、真菌の病原性のメカニズムの知識を進めるために有用なツールとなり、人間のマクロファージとの通性細胞内のヒトの真菌病原体のカンジダ·グラブラタの相互作用を研究するためのin vitroでの細胞培養モデル系を確立するためのプロトコルの概要を説明します。

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

establishment-an-vitro-system-to-study-intracellular-behavior-candida

Research

JoVE Journal

Immunology and Infection

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

11.5K ビュー.  Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India. 現在の記事は、真菌の病原性のメカニズムの知識を進めるために有用なツールとなり、人間のマクロファージとの通性細胞内のヒトの真菌病原体のカンジダ·グラブラタの相互作用を研究するためのin vitroでの細胞培養モデル系を確立するためのプロトコルの概要を説明します。 

ビデオ: Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

免疫・感染学

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

不定冠詞<em&gt;インビトロ</emヒトマクロファージの分化と偏光の不均一性を研究するために&gt;モデル

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

ライブマクロファージにおけるファゴリソソーム生合成の研究

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

ヌクレオによるヒトTHP-1マクロファージの高効率トランスフェクション

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

細胞内細菌によって遠藤-リソソームシステムの再構築を検討するために、蛍光ナノ粒子の応用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

誘導発現系を適用すると、細胞内シグナル伝達と細菌の病原性因子の干渉を研究するために、

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

インビトロ転写mRNAによる一次マクロファージの高効率トランスフェクション

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...