The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H +キリングセレクション<ol><li>細胞株<ol><li>安定的にNHE欠損細胞をトランスフェクト（ 例えば 、CCL39由来PS120細胞株16）線維芽細胞株で効率的なトランスフェクション収率及び選択を生成する哺乳動物発現ベクターおよびトランスフェクション方法の任意の組み合わせを使用して。 注：長年、リン酸カルシウム沈殿19が良いトランスフェクション収率で使用された。これは、リポフェクタミン2000などの市販の試薬によって、より最近で置換された、トランスフェクションは、製造業者の説明書に従って行われる。 </li></ol></li><li>ソリューション<ol><li>以下のように説明する3つの滅菌溶液を準備します。ろ過（0.22μm）とすることで、これらのソリューションを滅菌する。 <ol><li> 50mMのNH 4 Clを、70 mMの塩化コリン、5mMの塩化カリウム、1mMのMgCl 2、2のCaCl 2、5mMのglのからなるNH 4 +のロード液（pH7.4）を調製ucoseとpH7.4の15mMのMOPS。 </li><li> 120 mMの塩化コリン、5のKCl、1mMのMgCl 2、2のCaCl 2、pH7.0の5 mMグルコースおよび15 mMのHEPESから成るすすぎ液（pH 7.0）を準備します。 </li><li> pH7.4の120mMのNaCl、5mMのKCl、1mMのMgCl 2、2のCaCl 2、5mMグルコースおよび15mMのHEPESからなる回収液（pH7.4）を調製。 </li></ol></li></ol></li><li>選択を開始する前に、外部タンクのない追加のCO 2と共に37℃で使用することができる細胞培養インキュベーターを得る（CO 2と呼ばれる- 「フリー」インキュベーター）、5％とCO 2を損なうことがバッファリングをもたらす酸性化。約2×10 8細胞（典型的には約20コンフルエント100mmのプレート）に関心までのNHEを発現する細胞株を増幅する。 </li><li> H +手続きを殺す： <ol><li> 3で、ローディング溶液中で1時間関心の細胞内NHEを発現する細胞をインキュベートCO 2を含まないインキュベーターで7°C。 </li><li>ローディング溶液を吸引した後、上記の-リンス溶液中で二度、それらをすすぐ。酸性化が生成されます急なNH 4 +勾配の作成 ​​を損なう残留細胞外NH 4 Cl のを排除するために効率的にこの手順を実行します。 </li><li>吸引によるすすぎ媒体を除去し、CO 2フリーのインキュベーター中で37℃で再び回復ソリューションで1時間細胞をインキュベート。 </li><li>定期的な培養液（7.5％FCSを含む一般的にDMEM）を使用してリカバリメディアを交換し、（5％CO 2、95％空気の加湿雰囲気中で37℃）、標準的な培養条件で細胞を増殖させる。 </li></ol></li><li>安定なクローンが出現するまで、週2回の選択のこのサイクルを繰り返します。 注：細胞培養​​および選択条件に関する特別な注意を払ってください。仕事の月数がoになりやすい任意の汚染によって台無しにすることができます文化と繰り返し、細胞操作の長い期間の後ccur。 <ol><li>ここでは、クローンを増幅し、個別にさらにセクション2-3で説明し、それらを特徴づける、または特性化の前に細胞集団を生成するために、それらを一緒にプールするかどうかを選択します。 </li></ol></li><li>カウンターを選択する親の酸感受性表現型（ 図1B）に復帰した可能性があるものをすることによって酸性化耐性細胞を保持するために臨時の選択（約2週間に1回）を適用します。 </li></ol> 2.細胞内pH測定 2.1）蛍光性pHイメージング<ol><li> 490 nmで励起したときのpH感受性であり、製造者のプロトコルに従って、445 nmの等吸収点を有しているレシオメトリックpH感受性蛍光色素BCECF / AMを使用します。 <ol><li>また、製造者のプロトコルに従って他のpH感受性プローブ​​を、使用しています。 </li></ol></li><li> CON設定された撮影に使用します高感度ビデオカメラに接続された倒立顕微鏡のsisting。 450nmの励起に適した石英中性密度フィルタと対に490nmの狭帯域干渉フィルタと、このセットを装備する。 <ol><li>可能であれば比も両方λ1および/またはλ2のために、どちらが飽和または低すぎる設定されている基底蛍光値を避けるため1に近づくように、λ1とλ2のセットアップ同等の蛍光値にフィルタや照明条件の適切なセットを使用します。これは、その後、細胞内pHが変化しているものの、比率は検出されていない重要なアーティファクトをもたらすことができる。 注：このシステムは、適切な発光波長（BCECFための535 nm）における急速な灌流、タイムラプス2上記の波長で励起し、取得を有効にする必要があります。ソフトウェアは自動データ収集と保存を可能にしてシステムを装備。 </li></ol></li></ol> NHEフォワード活動の2.2）測定（の押出サイトゾルからのプロトン） <ol><li> NH 4 +のロード溶液中で1時間のインキュベーションによって細胞を酸性CO 2の非存在下で（ステップ1.4参照）。 </li><li>最後の5分間溶液にBCECF-AM（5μM最終濃度）を追加した後、細胞外のプローブを除去するためにNH 4 +ローディング溶液でそれをすすいでください。 </li><li>顕微鏡で細胞をマウントし、安定したベースラインを記録するために複数の画像を取る。リンス液（上記参照）を灌流する。これは、酸性化に対応する蛍光の低下をもたらす。 </li><li> pHが安定した後、リチウム+ / H +のNa + / H +またはK + / H +交換によって媒介されるpH回復速度を測定するために、下記の溶液のいずれかを灌流。各溶液をH +交換のために試験される潜在的結合カチオンを含んでいる。それらの一つは、輸送される場合、これはcorrespます495 nmでの蛍光増加をもたらす二の細胞内のpHの昇給へ： pH7.4の120mMのLiClを、5mMのKClを、1mMのMgCl 2、2のCaCl 2、5mMグルコースおよび15mMのHEPES pH7.4で120mMのNaCl、5mMのKCl、1mMのMgCl 2、2のCaCl 2、5mMグルコースおよび15mMのHEPES pH7.4での125mMのKCl、1mMのMgCl 2、2のCaCl 2、5mMグルコースおよび15mMのHEPES </li></ol> NHEリバース活動の2.3）測定注：ここでの原則は、膜貫通イオン性勾配を反転させることである。 <ol><li>測定前に、興味のある細胞内カチオンで細胞をロードする（下記参照）。 </li><li> （ステップ2.2.2を参照）BCECF / AMで5分間それらをインキュベートする。それらをすすぎ、ビデオ顕微鏡にマウント。 </li><li>ローディング溶液を灌流し、安定なベースラインを記録するために複数の画像を撮る。 </li><li>ベースラインが安定した後、画像セルが120メートルを含む細胞外酸性媒体で灌流されたpH変化Mの塩化コリン、5mMのKClを、1mMのMgCl 2、2のCaCl 2、pH6.5の5 mMグルコースおよび15 mMのMES。 </li><li> LiClをローディングのため、pH7.4の120mMのLiClを、5のKCl、1mMのMgCl 2、2のCaCl 2、5mMグルコースと15mMのHEPESからなる溶液中で2時間細胞をインキュベート。原子吸光分光法によって測定された細胞内のリチウムは、約60mMの値が得られる。 </li><li>のNa +をロードするために、1mMのウアバインの存在下で、pH7.4の120mMのNaCl、5mMのKCl、1mMのMgCl 2、2のCaCl 2、5mMグルコースおよび15mMのHEPESからなる溶液中で2時間細胞をインキュベートのNa / K ATPアーゼをブロックする。これらの条件において、細胞内のNa +濃度は、40mMの範囲である。 注：細胞質ゾルK +濃度が140 mMの範囲にあり、外側に指向K +勾配を達成するのは容易であるようにK +ローディングが不要である。 </li></ol> 2.4）データの処理と校正： 注：このステップでは、順方向および逆方向の測定値の両方に適用されます。 <ol><li>各実験の最後に、140ミリモルのKCl、20mMのHEPESおよび6.5と7.4の間のpH値に調整し、5μMナイジェリシンの溶液で細胞を灌流する。 </li><li>テキスト形式の下での画像（強度レベルを表すグレーレベル）と輸出から蛍光測定などのデータを収集し、スプレッドシートプログラムを用いて以下に説明するように扱う。 </li><li>個々のセルまたは次の式を使用して、関心領域のための細胞内pH値を計算する。 でpH = pKaは+（ログ（R-Rminを）/（R 最大 -R））×F 分（λ2）/ F MAX（λ2） </li><li> Fの分（λ2）/ FのMA×（λ2）実験を通して450 nmの蛍光の減少、その結果、プローブの漂白や漏れがあった場合の比率を使用してください。しかし、これは非常にまれにDESで使用される条件で観察されないcribed実験。 </li><li>較正データは、各pH測定の終了時に存在するように、対応する計算値を較正のために使用されるpH値と異なるかどうかをチェックする。それが事実である場合、次の手順を使用してキャリブレーションへのすべての実験的に決定されたpH値を調整します。 <ol><li>以下の商（Q、キャリブレーション値の間で測定された値/差と差）を計算します。計算値は、対応するキャリブレーション値と等しくなるように加算または減算することによって最終的な調整を行いますQ.によって全データセットを掛けます。 </li></ol></li></ol>ファストLi +の取り込みによるNHE7の初期速度の3測定<ol><li>マルチウェルプレート上の種子細胞（6ウェル-24）および上記または20に記載の交互ナイジェリシン/ウシ血清アルブミン（BSA）酸性記載NH 4 +ローディング技法のいずれかを使用して酸性化する。 </li><li&gt;の所望の量のために、上述したリンス液を用いて急速に二回、プレートを洗浄した後、（若干のKm値以下）1〜10 mMのリチウムを含有する取り込み溶液中で細胞をインキュベートし、カチオンおよび/または関心のある阻害剤の所望の濃度時間の。 </li><li>トランスポートが初期速度条件で動作することを保証するために短いと一貫性の取り込み期間（通常は1分以下）を維持します。また、取り込みのために使用されるすべてのソリューションが等張であることを確認してください。 </li><li>取り込み時間の終わりに、慎重に取り込み培地を除去し、氷冷リン酸緩衝食塩水（PBS）で細胞を4回洗浄する。 （そのうち4が理想的であるために10秒未満）リチウム流出を防ぐために可能な限り速く、これらの洗浄を行う。 </li><li>溶解する25％の硝酸のセル。 25％の硝酸のウェル当たり250μlのを適用し、それがピペットチップの端部と各ウェルを廃棄し、新しいミシガン州全体の井戸サスペンションを転送した後、少なくとも1時間放置crocentrifugeチューブ。 </li><li>細胞破片を除去するために15000 XG（室温）で5分間、それらを遠心分離、1.5ミリリットルマイクロ遠心チューブにライセートを転送します。 </li><li>原子吸光分光法21により上清のリチウム含有量を測定します。 注：異なる企業がリチウム中空陰極ランプを装備する必要は原子吸光分光計を提供する。これらのマシンは非常に正確なだけでなく、非常に繊細であるように、メーカーの指示に従ってください。 </li></ol>

<ol>
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The authors have nothing to disclose.

このプロトコルは、部位特異的突然変異誘発によって、その一次配列を変更することなく、原形質膜で細胞内のNa + / H +交換体を発現する細胞を選択する方法について説明します。これらの交換は今特徴付けることができる。 この方法は、原形質膜小胞でのNa + / H +交換体を発現する細胞株を選択するために、細胞内のプロトンの細胞毒性?...

ほとんどの細胞内区画は、タンパク質またはホルモンの成熟、人身売買、リサイクル、神経伝達物質の負荷のために重要なパラメータである酸性の管腔のpHを、表示します。これは、サイトゾルおよび小胞の内容の間のpH勾配が小胞のClC塩化物トランスポーター2に結合され、液胞型H + -ATPアーゼ1によって生成されることが示されている。ノックアウト（KO）マウスとヒト患者の両方で、これらのトランスポーターの重要性は、その遺伝子3-6の突然変異によって引き起こさ重い表現型によって強調されています。 ナトリウム-水素交換体SLC9Aファミリーのメンバー、またのNa + / H +交換体のためNHEsと呼ばれるが、細胞内pHと細胞容積調節において、ならびに上皮を横切る酸-塩基当量のベクトル輸送における重要なエフェクターであることが示されている。原形質膜NHEs、3つの高度に保存されのNa + / H +交換体、NHE 6,7に加えおよび9はトランスゴルジ網および初期エンドソーム7で表されている。それらの遺伝子の変異は、アンジェルマン様またはクリスチャンソン症候群8-9、家族ベースの自閉症10及び注意欠陥多動性障害11-12でリンクされている。これらの交換器はまた、アルツハイマー病感受性13とX連鎖精神遅滞隣接遺伝子症候群14などの神経変性の問題に関わってきた。まとめると、これらの研究は、脳の発達および/または機能のこれらの細胞内NHEsの重要性を強調している。 これらの交換器の細胞内局在化は、それらのイオン選択性、搬送方向、速度論的パラメータ、及び規制の正確な測定を妨げる。細胞内区画において発現すべてのトランスポーターの場合のように、それらの生化学的活性を評価するために、したがって、完全にそれらの生理学的役割とmechanを理解することは極めて困難である彼らの病理学的な意味を根底にあるISMS。高い細胞質ゾルK +濃度に基づいて、最も一般的に受け入れられて仮説は、彼らがK +結合したプロトン排出トランスポーターとして働いていたということでした。それは定常状態の水疱性pHを維持するために、V-ATPアーゼによってプロトンポンプを相殺できるようなプロトンリークの存在が仮定されていた。この視覚資料の目的は、それらの形質膜にこのような小胞輸送体を発現する細胞株の遺伝的選択を可能にする、および（ii）これらのトランスポーターの機能を測定するために、二つの独立したアプローチを示すための方法を実証するためには、（i）である。 30年前、PouysségurとフランキはNHEファミリー15のメンバーの分子クローニングおよび特性を有効に遺伝的アプローチを開拓してきた。これは、スクリーニング法として、細胞内のプロトンの毒性に基づいていた。最初のステップは、欠損細胞株を得ることであった任意のNa + / H +交換において、このトランスポーターの可逆性を用いて、原形質膜で発現する。線維芽細胞は、（CCL39細胞株）のNa +またはLi +でプリロードし、次いで2時間、酸性細胞外培地（pH6.5）中に入れた。これは、機能のNa + / H +交換体を発現する細胞の死およびアンチポーター欠損細胞（PS120細胞系）16の選択につながった。重炭酸塩を含まない培地中で培養した場合、これらの細胞は、急性細胞内酸性化に非常に敏感である。そのような細胞は、急性細胞acidificationsに提出された場合、結果として、原形質膜における任意の機能プロトン流出メカニズムの発現が正に（17を参照）が選択される。このような酸性化技術は、売買欠陥が原形質膜でWT細胞NHEsの強制発現を可能とする細胞株を単離することができる。 真核生物のNa + / Hとして+交換器は、彼らがチャンネルを測定するために大きな成功を収めて使用されている電気生理学的ア ​​プローチによって測定可能ではないが、電気的に中性である。この原稿は、したがって、細胞内pH測定及びリチウム取り込みの迅速な動態によって、この交換器の活性を測定する方法を示します。基礎となる概念が同じであるように、選択部のために開発プロセスの多くは、機能の測定のために直接使用されることに注意することは興味深い。 興味深いことに、我々は、この原稿に記載されたアプローチを使用して選択された細胞株に存在するトラフィッキング欠陥は、水疱性カリウムチャンネルTWIK1 18のように、原形質膜における他の小胞のタンパク質のより大きな発現をもたらすことを観察した。これは、小胞の膜貫通型タンパク質のための一般的なリテンション不良メカニズムの選択に向かって指摘している。したがって、この選択手順と、それが05を生成する細胞細胞内コンパートメントの膜タンパク質に取り組んで科学的なコミュニティのための有望なツールを構成している。同様にここで紹介する測定技術は、他の非起電性トランスポーターを研究するための適用可能な場合があります。

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard cell culture equipement</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Used for H+ killing selection composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator with CO2</td>
			<td style="width:119px;">Sanyo</td>
			<td style="width:119px;">MCO15A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator without CO2</td>
			<td style="width:119px;">Heraeus instrument</td>
			<td style="width:119px;">BB6220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laminar flow hood PSM1200NF</td>
			<td style="width:119px;">Fisher&#160;</td>
			<td style="width:119px;">52010120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">D5796</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS gold</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:119px;">A15-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicilin/streptomycin</td>
			<td style="width:119px;">PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 10X</td>
			<td style="width:119px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic Absorption spectrometer with Zeeman furnace system</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:119px;">ICE 3500 GFZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiCl</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">L4408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric Acid</td>
			<td style="width:119px;">sigma</td>
			<td align="right" style="width:119px;">438073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescence videomicroscopy set</td>
			<td style="width:119px;">Leica</td>
			<td style="width:119px;"></td>
			<td>Composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted automated microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:119px;">DMI6000B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microscope stand</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888906</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888911</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11505180</td>
			<td></td>
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			<td align="right" style="width:119px;">11888377</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">incident fluorescence</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
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			<td align="right" style="width:119px;">11504166</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">motor bracket</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11505234</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521505</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11522106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED transmission light&#160;</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">8097321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">8102034</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">motorized plate</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11522068</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11531172</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521734</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521719</td>
			<td></td>
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		<tr height="21">
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			<td></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera output</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11507807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888258</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11541510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">images acquisition/analysis software</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optics</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11506507</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506243</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fluorescence Xenon lamp</td>
			<td style="width:119px;">leica</td>
			<td style="width:119px;">DMI6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera</td>
			<td style="width:119px;">hamamatsu</td>
			<td align="right" style="width:119px;">8100601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">metafluor/Mmfluor software</td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11640905</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH sensitive probe, BCECF-AM</td>
			<td style="width:119px;">life technologies</td>
			<td style="width:119px;">B1170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nigericin</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">N7143</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

エンドソームの酸性化は、タンパク質のリサイクルと劣化、受容体の脱感作、およびシナプス小胞における神経伝達物質の負荷などのプロセス、幅広いするために重要である。この酸性化のClC塩化物輸送体に結合されたプロトンATPアーゼによって媒介されることが記載されている。電気的中性のプロトン輸送体を高度に保存された、のNa + / H +交換体（NHE）6、7及び9は、これらのコンパートメントにおいて発現される。それらの遺伝子の変異は、人間の認知と神経変性疾患にリンクされています。それらの細胞内局在化は、詳細な機能解析を妨げているように逆説的に、彼らの役割は、とらえどころのないまま。この原稿は、この問題を解決する方法を示している。これは、原形質膜で細胞内NHEsを保持することにより急性のサイトゾル酸性化の生存可能な変異体細胞系の選択から成る。次に、イオン選択性および活性を測定するために二つの相補的なプロトコルを示している（i）1種の蛍光ビデオ顕微鏡を用いて細胞内pH測定値に基づいて、および（ii）1つのリチウムの取り込みの迅速な動態に基づくこ​​れらの交換器。そのようなプロトコルは、他の非起電性輸送を測定するために外挿することができる。さらに、ここで提示選択手順は、細胞内の保持不良表現型を有する細胞を生成する。従って、これらの細胞は、原形質膜における他の小胞の膜タンパク質を発現する。ここに示された実験戦略は、したがって、その後の選択のために使用される小胞のNa + / H +の交換とともに原形質膜で発現される他の細胞内タンパク質を研究するための潜在的に強力なツールを構成することができる。

選択： 図1Aに示すように、選択を殺すH +は 、アンモニウム弱塩基の拡散に基づいている。細胞内のpHに弱塩基および酸拡散の効果がウォルターホウ素及び共同研究者22によって開拓されました。ポジティブ遺伝子選択のための致命的な酸性化を生成するために、この現象を使用するには、エレガントなアイデアは、その後ジャック·Pouyssé...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

ナの機能的特徴<sup&gt; +</sup&gt; / H<sup&gt; +</sup細胞膜でそれらを表現するためにプロトン殺す選択を使用した細胞内区画の&gt;交換器

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Research

JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K ビュー.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

ビデオ: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

アストロサイトの細胞膜とグローバル細胞内カルシウム動態の近くの測定

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

生物学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

フローサイトメトリーによる細胞膜へのGLUT4転の定量的測定

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

内因性野生型BRCA1を発現する細胞を用いて相同組換えにBRCA1の変異の影響を識別

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

蛍光タンパク質を発現する生きたマウスでのイメージング細胞内の構造物の生体顕微鏡

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

セルベースの​​L-グルタチオン保護アッセイ細胞膜タンパク質のエンドサイトーシスとリサイクルを研究するために、

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFMによる画像形成細胞膜変形は

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...