インペリアルカレッジにおけるin vivoイメージング施設で MRCによって資金を供給された。

<ol>
	<li>Chang, M. H., Cirillo, S. L., Cirillo, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Luciferase+to+Image+Bacterial+Infections+in+Mice.">Using Luciferase to Image Bacterial Infections in Mice.</a> J. Vis. Exp. (48), e2547 (2011).</li><li>Wiles, S., Pickard, K. M., Peng, K., MacDonald, T. T., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+the+murine+pathogen+Citrobacter+rodentium.">In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium.</a> Infect Immun. 74, 5391-5396 (2006).</li><li>Wiles, S., 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=Organ+specificity,+colonization+and+clearance+dynamics+in+vivo+following+oral+challenges+with+the+murine+pathogen+Citrobacter+rodentium.">Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium.</a> Cell Microbiol. 6, 963-972 (2004).</li><li>Wiles, S., Dougan, G., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+a+'hyperinfectious'+bacterial+state+after+passage+of+Citrobacter+rodentium+through+the+host+gastrointestinal+tract.">Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract.</a> Cell Microbiol. 7, 1163-1172 (2005).</li><li>Fanning, S., 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=Bifidobacterial+surface-exopolysaccharide+facilitates+commensal-host+interaction+through+immune+modulation+and+pathogen+protection.">Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 2108-2113 (2012).</li><li>Doyle, T. C., Burns, S. M., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+integrated+studies+of+infection.">In vivo bioluminescence imaging for integrated studies of infection.</a> Cell Microbiol. 6, 303-317 (2004).</li><li>Collins, J. W., 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=Pre-treatment+with+Bifidobacterium+breve+UCC2003+modulates+Citrobacter+rodentium-induced+colonic+inflammation+and+organ+specificity+of+infection.">Pre-treatment with Bifidobacterium breve UCC2003 modulates Citrobacter rodentium-induced colonic inflammation and organ specificity of infection.</a> Microbiology. , 10-1099 (2012).</li><li>Contag, C. 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol. Microbiol. 18, 593-603 (1995).</li><li>Hardy, J., 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=Extracellular+replication+of+Listeria+monocytogenes+in+the+murine+gall+bladder.">Extracellular replication of Listeria monocytogenes in the murine gall bladder.</a> Science. 303, 851-853 (2004).</li><li>Szittner, R., Meighen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence,+expression,+and+properties+of+luciferase+coded+by+lux+genes+from+a+terrestrial+bacterium.">Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium.</a> J. Biol. Chem. 265, 16581-16587 (1990).</li><li>Andreu, N., Fletcher, T., Krishnan, N., Wiles, S., Robertson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+antituberculosis+drug+activity+in+vitro+and+in+macrophages+using+bioluminescence.">Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.</a> J. Antimicrob. Chemother. 67, 404-414 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Cronin, M., 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=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Mundy, R., MacDonald, T. T., Dougan, G., Frankel, G., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium+of+mice+and+man.">Citrobacter rodentium of mice and man.</a> Cell Microbiol. 7, 1697-1706 (2005).</li><li>Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+skin+pigmentation+in+C57BL/6+mice+affect+optical+bioluminescence+quantitation.">Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.</a> Mol. Imaging Biol. 13, 1114-1123 (2011).</li><li>Wiles, S., Crepin, V. F., Childs, G., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biophotonic+imaging+as+a+training+aid+for+administration+of+substances+in+laboratory+rodents.">Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents.</a> Lab Anim. 41, 321-328 (2007).</li><li>Crepin, V. F., 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=Dissecting+the+role+of+the+Tir:Nck+and+Tir:IRTKS/IRSp53+signalling+pathways+in+vivo.">Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.</a> Mol. Microbiol. 75, 308-323 (2010).</li><li>Willekens, I., 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=Evaluation+of+the+radiation+dose+in+micro-CT+with+optimization+of+the+scan+protocol.">Evaluation of the radiation dose in micro-CT with optimization of the scan protocol.</a> ContrastMedia Mol. Imaging. 5, 201-207 (2010).</li><li>Boone, J. M., Velazquez, O., Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-animal+X-ray+dose+from+micro-CT.">Small-animal X-ray dose from micro-CT.</a> Mol Imaging. 3, 149-158 (2004).</li><li>Sato, F., Sasaki, S., Kawashima, N., Chino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+effects+of+whole+or+partial+body+x-irradiation+on+mice:+life+shortening.">Late effects of whole or partial body x-irradiation on mice: life shortening.</a> Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 39, 607-615 (1981).</li><li>Kohn, H. I., Kallman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+strain+on+acute+x-ray+lethality+in+the+mouse.+II.+Recovery+rate+studies.">The influence of strain on acute x-ray lethality in the mouse. II. Recovery rate studies.</a> Radiat. Res. 6, 329-338 (1957).</li></ol>

この記事への生産とフリーアクセスは、パーキンエルマーが主催しています。 著者はケビン·P、フランシス、ジェフMeganckとChaincy郭はキャリパー·パーキンエルマー社の従業員です。 全ての動物実験は、動物科学手続法1986に従って実施され、地元の倫理審査委員会によって承認された。

細菌感染の4D映画は、迅速かつ容易にマルチモダリティイメージング、大量のデータを視覚化し、解釈するための有用なツールを提供しています。この手法は、感染症は、個々のマウスを介して拡散し、縦断的研究7時にホストまたは細菌遺伝子または特定の介入戦略の効果細菌負荷、流通、ローカライズの方法削除を調査するために使用する方法の詳細な分析を容易にします。これらのビデオにも便利教材や国民に情報発信の手段を提供します。 DLIT-μCTイメージングと感染の4Dのビデオをコンパイルする機能から得られたデータの品質に影響を与える可能性があり、このプロトコルにはいくつかの重要なステップがあります。このプロトコルの最も重要な部分は、C.を有するマウスの正常かつ均質な感染症であるrodentium。これは、研究のために使用されるマウスは18〜20グラムの間で、そのあることが不可欠であるbacteリアル接種は、新たに調製し、約5×10 9 cfuのは、前述2,3に記載されている。以前はマウスの感染に、それは接種はスペクトルCTを使って、一度接種が準備されている生物であることを確認することが重要であるそれぞれのマウスがマウスが同じような感染量を受け取ることを保証するために強制経口投与する前に、それが継続的に均質化されている必要があります。マウスのDLIT-μCTイメージングは​​リビングイメージ4.3.1ソフトウェアの自動露出機能が自動的にうまくノイズの上になるように信号用に最適化された撮像パラメータを決定するように最適化されています。ただし、自動露光機能が手順で説明したように変更する必要がユーザ設定及びパラメータに依存している。これを怠ると、自動露出のためのスペクトラムCTの工場出荷時の設定は、撮像腫瘍が発現するためにプログラムされると、感染の明らかな進行をもたらさないことを集めた光子の数が少ないと悪いイメージになりますホタルルシフェラーゼ。復元は、560から620 nmのシミュレートと測定データとの間の最良の合意を与える使用して実行し、そのため、復興に含めるより信頼性の高いデータである。 DLIT-μCTの使用に制限はμCTスキャンからの電離放射線は、縦断的研究18の累積である亜致死放射線損傷の原因となります。亜致死放射線露光は、DNA損傷、および内臓19におけるアポトーシスを引き起こす免疫応答を弱めることができる。電離放射線のためのLD 30分の50はマウスのマウス系統や年齢に応じて、5〜7 Gyの間で18,20,21を使用され、超過した場合に最終的に、累積致死放射線損傷は、死を引き起こす可能性があります。包括的な原則は、保守的に線量を推定することであるため、電離放射線の分子損傷のいくつかは、癒すことができますが、これは一般的に研究計画では占めていません。その代わりに、目的はずっとベルとして滞在することですできるだけOWはこれらの制限はまだ研究の目標を達成しながら。これは、感染に対する正常な免疫応答のこの研究において特に重要である、画像化、およびトランスジェニック動物を免疫構成され、又は頻繁に感染しているという事実の周波数は、電離放射線の影響をより受けやすいかもしれない。 感染の4Dムービーを生成する実験を計画するとき、それは実験の長さを考慮することが重要である、μCT数が使用されているマウス系統に対して電離放射線のために、この期間中に必要とLD 30分の50スキャンします。これは細菌の検出限界とイメージング時間に影響を与えるとしてDLIT-μCTに別の潜在的な制限は、使用されている菌株内レポーター発現の強さです。 BLIのために以前に実証されるようにこれは非常に、研究者が完全に病原性である菌株を検証し使用することをお勧めしますが、最大のルクスオペロンの発現のために最適化され2,3。 4Dイメージングの現在の設計の一つの注意点は、それぞれのムービーが異なる光子スケーリングを持つ個々のDLIT-μCTスキャンで構成されているということです。 BL病巣の局在、またはその強度に変化は微妙である場合、これは解釈が画像を困難にすることができたり、複数の弱巣に囲まれた1激しいBLフォーカスが存在する場合。したがって、長手方向の視覚化のために、それは時間ポイント間のカラーバーの一貫性を保つことが重要である。 感染の4D映画の概念は、任意の適切に標識された細菌性病原体に適用することができる。この技術の今後の展開は、感染に対する宿主応答を調査する赤外線プローブ近い蛍光生物発光細菌性病原体および注射の組み合わせを使用して、感染に対する宿主応答の調査を容易にするために、蛍光イメージング断層撮影（FLIT）ならびにDLITを使用することを目指します。さらにこれに、このプロトコルで我々だけ感染の4Dムービーを作成するための生物発光細菌の使用を記載している。しかし、いくつかの例では、生物発光レポーター、感染時にホスト遺伝子を調査するために使用することができるので、IRFPでタグ付けされ、例えば、蛍光標識された細菌を使用する必要があるかもしれない。重要なのは、DLIT / FLIT-μCTを組み合わせたマルチモダリティイメージングの使用は、私たちは非侵襲的に削減、洗練、および科学研究における動物の使用の交換に大きく貢献する細菌感染症、間に複数のパラメータを調べることができますNC3Rのイニシアチブ（に概説されているよう<a href="http://www.nc3rs.org.uk/" target="_blank">http://www.nc3rs.org.uk/</a> ）。

特に小さな動物モデルは、これらのマウスを利用して、日常的に細菌の病原性を調査するために、又は抗生物質、プロバイオティクス、プレバイオティクスおよびワクチン1-7などの感染のために介入戦略をテストするために使用される。小動物の感染症からの主な実験的な読み出しは病原体負荷、感染症の空間的および時間的局在、および感染した生物の免疫応答への変更である生体内光学イメージングでは感染症研究のための貴重なツールであり、複数の監視に使用できますレポーター遺伝子を使用することにより実験的読み出し（ルシフェラーゼ、蛍光タンパク質、β-ラクタマーゼ、等）、蛍光色素、ナノ粒子またはタンパク質、生物学的プロセス、又は微生物6に標的化学発光プローブ。 生物発光イメージング（BLI）が病原性bacteにより、例えば、マウス、ラットなどの小動物のコロニー形成を監視するために使用される光学撮像モダリティであるRIA 3,6,8,9。マウスはそのようなPhotorhabdusのluminescensからルクス CDABEオペロンとしてルシフェラーゼを発現する組換え細菌に感染している。これらの細菌は、その後vivoイメージングシステム3,6,9 に基づいてCCDを使用して光生成を介して検出することができます。重要なのは、唯一の代謝的に活性な微生物は、生存細菌細胞は、この方法論10,11によって検出される、つまり生物発光（BL）である。 2D BLI使用して、BL源の位置は、信号が発せられる動物8の表面から推測される。 in vivoでの BL病巣の正確な解剖学的局在は対照的に、臓器3,6,9のex vivoでの分析によって決定されなければならない、合成3次元拡散光トモグラフィイメージング（DLIT）はBLの定量的な3次元再構成をコンパイルするために使用することができるソース12。 DLITが定義狭帯域通過光フィルタを用いて撮影したBL画像を収集することによって行われ、その後、拡散光トモグラフィー3次元再構成アルゴリズム1,7,12,13にそれらを入力する。 現在のところ、マルチモダリティイメージングはex vivoでの解析を必要とせずに、in vivoで生物発光病巣の真の非侵襲的な解剖学的局在を得ることができる唯一の方法論です。最近、我々はDLITプロバイオティクス細菌7と予防的治療後にシトロrodentium（C.rodentiumの ）植民地化の動態を評価するためにμCTイメージングと共同で登録された組み合わせを使用していました。C. rodentiumは病原性とenterhemorrhagic 大腸菌 14とモデル、人間の感染に使用されたマウス特有の腸病原体である。Cでrodentium感染は一般的に軽度の体重減少、下痢、偏Th1型免疫応答と結腸陰窩の過形成など、明確な病理学的変化、及び取り付け、控えめ病変のformatiに関連付けられている、大腸炎を引き起こす14。これに加えて、C. rodentiumの病因は、徹底的にこの細菌マルチモダリティイメージング3,4,7での使用に理想的なモデル微生物作り、十分に文書化されているC57BL/6JマウスにBLIとその植民地化のダイナミクスを使って研究されてきた。 このプロトコルは、単一のマルチモダリティイメージングプラットフォーム、IVISスペクトラムCT、および非侵襲的に、この感染症の真の力学的な関係を示すの4D映画の生成を使用して細菌感染の統合DLIT-μCTイメージングのための方法論を概説する最初のものです。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bioluminescent C. rodentium</td>
			<td>Frankel lab</td>
			<td>ICC180</td>
			<td>Wiles et al., 2004</td>
		</tr>
		<tr>
			<td>Veet</td>
			<td>Boots</td>
			<td>&nbsp;</td>
			<td>Optimal depilation time is 7 min. Depilation works better if the cream is rubbed in well.</td>
		</tr>
		<tr>
			<td>Isofluorane (100% v/v)</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medical Oxygen</td>
			<td>BOC Medical</td>
			<td>&nbsp;</td>
			<td>Size F Cylinder. Note: an appropriate regulator is required.</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Merck</td>
			<td>1.10285.0500</td>
			<td>25 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Merck</td>
			<td>1.10283.0500</td>
			<td>37 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Sigma (Fluka)</td>
			<td>60615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Falcon tubes</td>
			<td>BD (Falcon)</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Universals</td>
			<td>Corning (Gosselin)</td>
			<td>E5633-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD (Plastipak)</td>
			<td>300013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oral dosing needle (16G x 75 mm) curved</td>
			<td>Vet Tech</td>
			<td>DE005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microbanks (Cryovial)</td>
			<td>Pro-Lab Diagnostics</td>
			<td>PL.170/Y</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum CT</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>133577 Rev A/ Spectrum CT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6kVA UPS</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XGI-8 anesthesia system</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118918</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XAF-8 Anaesthesia filter charcoal</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118999/00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image v4.3.1 SP1</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benchtop shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 44</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

4d multimodality imaging of citrobacter rodentium infections in mice

このプロトコルは、縦方向に感染サイクルの4次元（4D）のムービーを生成するために統合されたμCT（DLIT-μCT）及びこのデータのその後の使用との複合3D拡散光イメージングトモグラフィーを用いた生物発光細菌感染を監視するために必要な手順の概要を説明します。 4D感染ムービーを開発し、IVISスペクトラムCTを用いた細菌感染症研究のためDLIT-μCTイメージングを検証するために、我々は生物発光C.感染を使用マウスで自己限定性大腸炎を引き起こすrodentium、。このプロトコルでは、生物発光C.によるマウスの感染を概説rodentium、毎日DLIT-μCTイメージングと8日間の糞便からの細菌の列挙によって植民地化の非侵襲モニタリング。 IVISスペクトラムCTの使用は、単一のイメージングプラットフォームを用いた光とμCTスキャンのシームレスな同時登録を容易にする。低用量μCTのモダリティは、マウスのイメージングを可能にする累積放射線からアーチファクトを引き起こすことなく、3Dで生物発光細菌の巣の詳細な解剖学的局在を提供する感染中の複数の時点、で。重要なのは、感染したマウスの4D映画がin vivoで細菌のコロニー形成のダイナミクスを監視するための強力な分析ツールを提供する。

5×10 9 cfuのC.とC57BL/6Jマウスの感染rodentiumはよく説明し、細菌感染モデルと自己限定胃腸感染の結果である日6-8感染後の間に3〜4週間2,14間続く ​​ピーク。感染症は、マウス免疫系によって腸管腔に限定されており、結果として、細菌が絶えず糞便中に流されている。 C.によるマウスの植民地化rodentiumは糞、または生物発光イメージング2,3から直接細菌の列挙によって非侵襲的に監視することができます。 本稿では、Cの 3Dと4Dのマルチモダリティイメージングを実行するための最適化されたプロトコルを概説感染中の細菌負荷およびローカリゼーションを監視するマウス内rodentium。 図1に示した結果が成功の4Dイメージングのために必要とされる制御を示す。それを阻止することが不可欠であるマウスの感染に先立ち使用される細菌の接種は、生物発光（ 図1A）であることを鉱山とその生物発光Cとマウスの強制経口投与後に信号は動物の胃の中ではなく、肺（ 図1B）16で観察できることrodentium、。 生物発光イメージングを使用して細菌のコロニー形成を監視することに加えて、それは糞便中の細菌数を定量化することをお勧めだ、胃腸粘膜の細菌定着の間接的な測定として使用される図2 8 C57BL6 / Jから取ら糞便中の典型的な細菌負荷を示しています。 〜5×10 9 CFU ICC180に感染し、約5×10 10 CFU / gであり、一日6-7どこで感染ピーク日まで2 PIから8日間のPI植民地の上昇のために監視されているマウス 以前の報告2,3,17に沿ったです。 評価の重要性を強調するにはティンシングルマウス、 図1b及び3と同様に、 ビデオ1での感染の広がりが C以下同じマウスから生成されています上述したようにrodentium感染 、。毎日DLIT-μCT、解剖学的基準としてスケルトンを使用して、このマウス内の生物発光細菌の空間分布を評価した。 図3は、少なくともPI 3キーの時点でICC180に感染したC57BL/6JマウスのDLIT-μCT再構成を示してい3日目PI小さな生物病巣は空間分布の変化が少ないと5日目PIによる生物発光強度の緩やかな増加を示す結腸で観察することができる。 7日目PIで私たちは生物発光の大幅な増加と結腸全体にわたる生物病巣の広がりを観察した。 ビデオ1日からICC180と1-8 PI 3D DLIT-μCT再建の集大成であり、Cを示しています日で細菌病巣は結腸全体をカバーする7 PIピーキングまで大腸内細菌の数字は拡大4-6 PI日から3日目のPIで大腸に広がる日1-2 PIの間に近位消化管内rodentium。 8日目PIのみで二つの異なる細菌の巣には、近位および遠位結腸に存在している。時系列順に各時点で完全な3D再構成を表示する機能は、ホスト病原体の相互作用を分析するための強力なツールを表し、同じデータセットの3D静止画より解釈が容​​易である。 <img alt="図1" src="/files/ftp_upload/50450/50450fig1.jpg" /> 図1の2次元生物発光イメージング）生物発光C. rodentium ICC180接種と接種200μlで強制経口投与後にB）マウス。アローヘッドは（&gt;）生物発光C.を示しrodentium </<html> em&gt;の胃である。 <img alt="図2" src="/files/ftp_upload/50450/50450fig2.jpg" /> 図2。C. rodentium植民ダイナミクス℃の定量8日間PIのため糞からrodentiumコロニー形成単位<img alt="図3" fo:content-width="3in" fo:src="/files/ftp_upload/50450/50450fig3highres.jpg" src="/files/ftp_upload/50450/50450fig3.jpg" /> 図3。生物発光Cの拡散光イメージングトモグラフィー-μCTスキャン3日目、5と7 PIアロー（&gt;） で監視1マウスからrodentium感染は結腸植民地化を示しています。 <a href="https://www.jove.com/files/ftp_upload/50450/50450fig3large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください</a> 。 ビデオ1。P :/ / www.jove.com/files/ftp_upload/50450/LAB_MEDIA_50450_Frankel_Video1.wmv &quot;ターゲット=&quot; _blank &quot;&gt;映像を表示するには、ここをクリックしてください。 日1から8までのPIを監視1マウスからC.rodentiumの感染の 4D映画

Watch this Scientific Journal Video about 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice at JoVE.com

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

マルチモダリティイメージングは​​、小さな動物モデルで細菌のコロニー形成を研究するための貴重なアプローチである。このプロトコルは、生物発光とマウスの感染症を概説<em&gt;シトロバクターrodentium</em&gt;との4Dムービーを作成するためにμCTイメージングと複合3D拡散光イメージングトモグラフィーを用いた細菌のコロニー形成の長手方向の監視<em&gt; C. rodentium</em&gt;感染。

の4Dのマルチモダリティイメージング<em&gt;シトロバクターrodentium</emマウスにおける&gt;感染症

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

4d-multimodality-imaging-of-citrobacter-rodentium-infections-in-mice

Research

JoVE Journal

Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

12.9K ビュー.  Imperial College London. マルチモダリティイメージングは​​、小さな動物モデルで細菌のコロニー形成を研究するための貴重なアプローチである。このプロトコルは、生物発光とマウスの感染症を概説シトロバクターrodentiumとの4Dムービーを作成するためにμCTイメージングと複合3D拡散光イメージングトモグラフィーを用いた細菌のコロニー形成の長手方向の監視

ビデオ: 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫・感染学

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

ザ<em&gt;シトロrodentium</em&gt;マウスモデル：感染性大腸炎に勉強病原体とホスト貢献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

生物発光イメージングを使用した多剤耐性アシネトバクターバウマニバーン感染症のための抗菌ブルーライトセラピーのインビボでの調査に

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

レポーター酵素蛍光を用いたマウスにおける結核菌をイメージング

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

上皮細胞の赤痢菌の感染時に Ca2 +応答をイメージング

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

二本鎖 RNA と否定的な感覚の RNA のウイルス感染におけるパターン認識受容体の共焦点レーザー顕微鏡

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

高スループット赤痢菌-特定の殺菌試験

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...