The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1感作マウス<ol><li> 20ミリグラム/ mlの最終濃度になるように1×PBS中のOVAを希釈することによってOVAストック溶液を調製する。クリオバイアル中にアリコートを下に凍結し、将来の使用のために-80℃で保存してください。 </li><li>室温に20 mg / mlのOVA株の一つ以上の分量を持参してください。キャップ5ミリリットルのポリスチレン丸底チューブに1 mg / mlでの最終濃度になるように1×PBS中OVAを希釈する。ボルテックスを簡単に混合する。 </li><li> 1の比率：中速に設定したボルテックスで1 mg / mlのOVAを含むポリスチレンチューブを押さえながら1を生成するために、チューブをボルテックスを続けながら、十分に均質化Imjectミョウバン（40 mg / ml）を滴下して、等量のを追加免疫原に対するミョウバン。 </li><li>渦にチューブ、テープ、チューブのキャップを置きます。最も低い設定で渦をオンにして、OVAを30分間ミョウバンに吸着することができます。 <ol><li>多くの時間は、OVA /ミョウバンを用意し、マウスを感作の間を通過させないようにしてください。感作は、OVA /ミョウバンpreparat直後に完了できない場合室温でのイオン、店舗OVA /ミョウバン。使用する前に1分間ボルテックスする。 </li></ol></li><li> OVA /ミョウバン混合物で1ミリリットルのインスリン注射器をロードします。 27ゲージの針を注射器に蓋を。 </li><li> OVA50μgのマウス当たりの用量及びミョウバン2mgのためのBALB / cマウスの腹腔にOVA /ミョウバンを100μlを注入。 </li><li>ネガティブ対照として、PBSに対するマウスの別のグループを感作。 OVAを省略して、上記と同様に、ミョウバンとPBSの1：1混合物を準備します。 </li><li> 3回の注射で合計3週間、週に一度、すべてのマウス（OVAおよびPBS対照）を感作する。最後の注射の後、地元のアナフィラキシーアッセイを行う前に少なくとも2週間待ちます。 </li></ol> 2ローカルアナフィラキシーアッセイ<ol><li>室温にOVA原液を持参してください。 （：1×PBS中のOVAの4希釈1）を1.5 mlマイクロチューブでは、PBSで5ミリグラム/ mlの最終濃度に20 mg / mlのOVAストックを希釈する。ボルテックスを簡単に混合する。 </li><li> &lt;RC 2を使用して、マウスを麻酔/ SUP&gt;齧歯類麻酔システム（または同等の気化したデリバリーシステム）。イソフルランでリザーバを入力し、O 2気流をオンにします。誘導チャンバ内にマウスを置き、麻酔を開始するために、5％イソフルランコントロールダイヤルを調整します。マウスが完全に麻酔をかけた後、移動性の欠如によって示される、深い麻酔を維持するために、3％イソフルランコントロールダイヤルを調整します。この手順の麻酔の目的は、動物を固定化することです。耳と尾静脈注射は動物に重大な痛みや苦痛を引き起こさない。 注：イソフルランは、人間の生殖系に悪影響を及ぼす可能性があります。廃ガスを中和するために換気の良い部屋で使用し、誘導室に掃気キャニスターを取り付けてください。イソフルランの使用中に妊娠している場合は、露出を最小限にするために、炭素フィルターマスクを着用してください。 </li><li> 31 G針でキャップされた3月10日のccインスリン注射器にロード5 mg / mlのOVAを10μlの。 10μlの別の3月10日のccインスリン注射器をロード1倍のPBS。 </li><li>解剖顕微鏡を使用して、OVA50μgののチャレンジ用量のために、麻酔したマウスの右耳にOVAを10μlを注入する。このよう20μgのような他の用量もまた、許容可能である。 <ol><li> 15ミリリットルコニカルチューブの周りにスコッチテープ（粘着面を上に）ラップ、注射のために耳を安定させるために。テープに右耳の背側を固定します。耳の二枚の葉の間に針のベベル側を上に挿入します。ゆっくりあらゆる血管をヒットしないように注意しながら、耳組織の中心にOVAを注入。 </li></ol></li><li>上記と同じ方法を使用して、陰性対照として左耳にPBS10μlを注入する。 </li><li> 3分待ちます。 <ol><li>この時間の間に、拘束具にマウスを配置し、血管を拡張するために加熱ランプ下または1分間の温水浴中で尾を保持する。 </li></ol></li><li> 27 G針でキャップされた1ミリリットルのインスリン注射器を使用して、ゆっくりとテールVに0.5％エバンスブルー色素を200μl注入マウスのEIN。全体を200μlのが投与される前に、急速な注入は、血管構造を破壊することができる。この場合は、尾の反対側に静脈内に染料の残りの容積を注入しようとする。 </li><li> 10分待ちます。 </li><li>マウスを安楽死させると、鉗子や外科用ハサミを使用して、両耳を削除します。 <ol><li>に圧力が耳の中央に注射部位に適用されていないことを確認して、鉗子で耳の端をつかむ。近くにカットして、できるだけ多くの耳組織を削除ではなくを通じて、耳の付け根で軟骨に存在する尾根。毛皮少量の切除された耳の上に存在してもよい。これは、アッセイには影響しません。 </li></ol></li><li>一定分量1.5mlのマイクロチューブにホルムアミド700μlの。耳ごとに1つのチューブ（マウスあたり2の管）を準備します。 注：ホルムアミドは、催奇形性、変異原性、および刺激物である。手袋、眼の保護、保護面を着用すること。取り扱いには妊娠中の女性は注意が必要ですsのホルムアミドは胎児毒性があります。 </li><li> 、ホルムアミド、チューブにキャップをし、63℃に設定し、ドライ熱浴中で一晩インキュベートし、耳を追加します。耳はまだインキュベーション後に、わずかに青い表示される場合があります。しかし、染料の大部分は、ホルムアミド中に抽出されます。 </li><li> 96ウェルプレートに、二重に、各サンプルを300μlを追加。それが試料中に存在する場合に毛皮を転送避ける。彼らは吸光度の読み取りに影響を与えるように、ウェル中に存在気泡がないことを確認します。 </li><li> 96ウェルプレートに、二重に、ホルムアミドを300μlを追加。これらのウェルはブランクとして使用されます。 </li><li> 620 nmの吸光度を測定するための分光光度計を使用してください。各サンプル読みからホルムアミド井戸を引きます。 注：すべての実験は制服サービス大学施設内動物管理使用委員会によって承認されたプロトコルの下で実施した。 </li></ol>

<ol>
	<li>Kojima, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+and+Bbasophils+are+selectively+activated+in+vitro+and+in+vivo+through+CD200R3+in+an+IgE-independent+manner.">Mast cells and Bbasophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner.</a> The Journal of Immunology. 179 (10), 7093-7100 (2007).</li><li>Parikh, S. A., Cho, S. H., Oh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+enzymes+in+mast+cell+granules+and+their+potential+role+in+allergic+rhinitis.">Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.</a> Current Allergy and Asthma Reports. 3 (3), 266-272 (2003).</li><li>Amin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+allergic+inflammation.">The role of mast cells in allergic inflammation.</a> Respiratory Medicine. 106 (1), 9-14 (2012).</li><li>Nakaya, M., Takeuchi, N., Kondo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+histamine+receptor+subtypes+in+human+inferior+turbinates.">Immunohistochemical localization of histamine receptor subtypes in human inferior turbinates.</a> Annals of Otology, Rhinology &amp; Laryngology. 113 (7), 552-557 (2004).</li><li>Ramsdell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Trypan+Blue+to+demonstrate+the+immediate+skin+reaction+in+rabbits+and+guinea+pigs.">The use of Trypan Blue to demonstrate the immediate skin reaction in rabbits and guinea pigs.</a> The Journal of Immunology. 15 (4), 305-311 (1928).</li><li>Chase, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+sensitization+of+animals+with+simple+chemical+compounds:+X.+Antibodies+inducing+immediate-type+skin+reactions.">Studies on the sensitization of animals with simple chemical compounds: X. Antibodies inducing immediate-type skin reactions.</a> The Journal of Experimental Medicine. 86 (6), 489-514 (1947).</li><li>Goose, J., Blair, A. M. J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+rat,+induced+with+two+homologous+reagin-like+antibodies+and+its+specific+inhibition+with+disodium+cromoglycate.">Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies and its specific inhibition with disodium cromoglycate.</a> Immunology. 16, 749-760 (1969).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+mouse.">Passive cutaneous anaphylaxis in the mouse.</a> The Journal of Immunology. 81 (4), 355-357 (1958).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+reactions+in+the+skin+of+experimental+animals+provoked+by+antibody-antigen+interactoin.">Immediate reactions in the skin of experimental animals provoked by antibody-antigen interactoin.</a> Progress in Allergy. 5, 459-508 (1958).</li><li>Akiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitiative+evaluation+of+passive+cutaneous+anaphylaxis+(PCA)+using+a+hand-held+spectrophotometer.">Quantitiative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.</a> Biological &amp; Pharmaceutical Bulletin. 19 (8), 1112-1114 (1996).</li><li>Teshima, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+spectrophotometric+analysis+of+passive+and+active+ear+cutaneous+anaphylaxis+in+the+mouse.">Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.</a> Toxicology Letters. 95 (2), 109-115 (1998).</li></ol>

The authors have nothing to disclose.

アレルギー性​​疾患の多数のマーカーが気道チャレンジの設定における気管支肺胞洗浄液中へ循環させるヒスタミン、Th2サイ​​トカインの産生、および細胞動員のレベルの変化を含む、アレルゲン攻撃後のアレルギー反応の強度を評価するために使用される。これらのパラメータの変化をモニタリングすること、アレルギー反応を研究するために重要であるが、生物学的マーカーは、常?...

I型過敏症は、肥満細胞および好塩基球の表面上のIgE抗原によって誘発される架橋によって媒介される。これは、細胞の脱顆粒およびヒスタミン、トリプターゼ、および血小板活性化因子2のような血管作動および炎症誘発性メディエーターの放出をもたらす。脱顆粒中に予め形成されたメディエータの放出に続いて、肥満細胞を合成し、プロスタグランジンおよびロイコトリエンを放出し、さらに血管透過3を増加させる。初期臨床反応が急速に発生し、「即時反応」と呼ばれている。皮膚では、膨疹·アンド·フレア応答は、抗原チャレンジの数分以内に容易に見ることができる。チャレンジの用量に応じて、それは数時間後に「後期応答」を観察することができる。後期相腫れ、組織2への局部的な浮腫および白血球動員によるものである。ヒスタミン、一般的に参加している主要なメディエーターであると考えられて即時アレルギー反応は、ヒスタミン受容体1（HR1）に作用して血管やヒスタミン受容体2（HR2）上で発現平滑筋上に発現する。複合効果は、炎症4の部位で血流および血管透過性を増加させる。 アレルギーの動物モデルのさまざまなアレルギー性​​喘息、全身性アナフィラキシーおよびアナフィラキシーの局所モデルを含むアレルギー性​​炎症に関与する機構を研究するために開発されてきた。静脈内の色素投与が出版物は1920年代5に戻って、この技術の出会い系を記述すると、ほぼ一世紀のための動物モデルでのローカライズされたアレルギー反応を測定するために使用されています。ウサギおよびモルモットは、即時型過敏反応をテストするために使用される第一の動物モデルであった、そして最も敏感な応答は、一般に、耳5,6で見出された。アッセイは、後に、ラット7およびマウス8で使用するために検証された。 歴史的に、実験方法のさまざまな前の色素の注入は、染料の注入に先立っての抗原の注射への抗原の注射、及び染料と抗原の同時注射を含む、使用されている。アレルギー反応を測定するための手段としての静脈内投与の染料は、それが受動的、能動測定するために使用され、受動的な反応5,9を逆にすることができるように汎用性の高いアッセイである。数多くの染料は、トリパンブルー、ポンタミンスカイブルー、エバンスブルー、チバガイギーブルー536、インドインク5,6,9を含むアレルギー反応を評価するために利用されてきた。 0.5％エバンスブルー溶液は、現在、皮膚にアレルギー反応を測定するために使用される標準的な染料である。 チャレンジに対するアナフィラキシー反応は一過性である。最大強度は10内に到達する-染料注入の15分であり、染料に関係なく9に使用される動物種の、チャレンジ後30分以上を投与された場合も反応は見られない。色素血管外遊出の定量化は、元あったLY青色色素7-9で示されるように膨疹の大きさを測定した。さらに、脱顆粒したマスト細胞の数は、反応部位から皮膚組織を切除し、7トルイジンブルーで染色することにより定量することができる。肥満細胞は、高親和性IgE受容体FcεRIとを発現している主ローカルな細胞集団であるように、マスト細胞脱顆粒は、多くの場合、皮膚、IgE媒介アレルギー応答のためのマーカーとして使用される。組織への色素血管外遊出を測定するための分光光度技術は1990年代にラット10およびマウス11における受動皮膚アナフィラキシー（PCA）のために開発された。 次のローカルアナフィラキシーアッセイプロトコルは、1。小島らから適応し、アレルギー反応を誘発するための抗原として鶏卵卵白アルブミン（OVA）を利用した。しかしながら、所望であれば、OVA以外の抗原を使用してもよい。アッセイは、その後、血管permeabilitの変化を監視するためにエバンスブルー色素を使用する細胞のIgE架橋およびヒスタミン放出をマストに起因し発生するyの。

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BALB/c Mice</td>
			<td style="width:167px;">The Jackson Laboratory</td>
			<td style="width:135px;">651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PBS pH 7.4</td>
			<td style="width:167px;">Quality Biologic</td>
			<td style="width:135px;">114-058-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ovalbumin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">A5503-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Imject Alum</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:135px;">77161</td>
			<td style="width:225px;">Mix thoroughly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Evans Blue Dye</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">E2129</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">295876</td>
			<td style="width:225px;">99%+ Spectrophotometric grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Isoflurane, USP</td>
			<td style="width:167px;">Phoenix</td>
			<td style="width:135px;">NDC 57319-474-06</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1cc Insulin syringes</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">329654</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3/10 cc Insulin syringe with 31G needle</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:135px;">NDC 100861</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27G Needles</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">305109</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Forceps</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">11000-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Surgical scissors</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">14070-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">5ml Polystyrene round-bottom tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352058</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1.5ml Microcentrifuge tubes</td>
			<td style="width:167px;">Medical Supply Partners</td>
			<td style="width:135px;">15-1151</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">15ml Conical tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352097</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flat-bottom 96 well plate</td>
			<td style="width:167px;">Costar</td>
			<td style="width:135px;">3590</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Scotch tape</td>
			<td style="width:167px;"></td>
			<td style="width:135px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">RC2 Rodent Anesthesia System</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:135px;">922100</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vortex Genie 2</td>
			<td style="width:167px;">Scientific Industries</td>
			<td style="width:135px;">SI-0236</td>
			<td style="width:225px;">Model G560 with 3 inch platform</td>
		</tr>
	</tbody>
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measuring local anaphylaxis in mice

アレルギー反応は、肥満細胞および好塩基球、および血管作用性のその後の放出および炎症誘発性メディエーターの活性化の結果である。感作された個体におけるアレルゲンへの暴露は、生命​​を脅かすアナフィラキシーにマイナー紅斑によって異なる臨床症状をもたらす可能性がある。研究室では、さまざまな動物モデルは、アレルギー反応を駆動するメカニズムを理解するために開発されている。本明細書では、局所的なアレルギー反応を定量化するために、血管透過性の変化を測定するための詳細な方法を記載している。ローカルアナフィラキシーアッセイは、最初に1920年代に報告された、と小島らによって公表さテクニックから適応されています。 2007年に1。このアッセイでは、OVAに感作したマウスは、車両と左耳にし、OVAでの右耳にチャレンジする。これは、エバンスブルー色素の静脈内注射が続く。十分エバンスブルーを注入した後、動物を中に浸出している染料を安楽死させている耳にホルムアミド中で一晩抽出した。抽出された色素の吸光度を分光光度計で定量化される。この方法は、確実に地元のアレルギー反応の視覚的および定量化可能な症状をもたらす。

成功した検定を受けた動物は、青色に見える皮膚や目を持つことになります。 PBS感作動物は、したがって、両方の耳が（ 図1A）白のままべきで、PBSまたはOVAチャレンジのいずれかに反応すべきではない。 OVA感作動物では、PBSチャレンジを受け取っ耳（左）は、注射部位の局所的な方法で完全に白か軽く青のどちらかである必要があります。 OVAチャレンジを受けた耳（右）は染?...

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

肥満細胞および好塩基球の活性化によって特徴付けられるアレルギー反応は、IgEの架橋および前炎症性メディエーターの放出によって駆動される。アレルギー反応の定量的評価は、アレルゲンチャレンジ後の血管透過性の変化を監視するためにエバンスブルー色素を用いることによって達成することができる。

マウスにおけるローカルアナフィラキシーの測定

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Research

JoVE Journal

Immunology and Infection

19.1K ビュー.  Uniformed Services University of the Health Sciences. 肥満細胞および好塩基球の活性化によって特徴付けられるアレルギー反応は、IgEの架橋および前炎症性メディエーターの放出によって駆動される。アレルギー反応の定量的評価は、アレルゲンチャレンジ後の血管透過性の変化を監視するためにエバンスブルー色素を用いることによって達成することができる。 

ビデオ: Measuring Local Anaphylaxis in Mice

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

マウスにおけるγHV68感染の測定

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

マウスのリンパ管内免疫療法やワクチン

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

変形能と赤血球による血液中赤血球の不均一性を測定

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酵素抗体法を用いたマウスにおける胸腺依存性および胸腺非依存性免疫グロブリン アイソタイプ反応の評価

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

パッシブ全身性アナフィラキシーと食品アレルギーの評価中に赤外線放射温度計を使用してマウスの体温測定

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

マウスにおけるアテローム性動脈硬化症の定量化

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

グルコース-6-リン酸イソメラーゼ(G6PI)誘発RAマウスにおけるiNKT細胞の導入免疫療法

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

フローサイトメトリーを用いたヒト白血球によるアスペルギルス・フミガトゥス・コニディアの食細胞症の測定

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...