1。 H.とマクロファージの感染カプスラーツム、C.アルビカンス <ol><li> 16感染前にHR、24ウェルプレート中で所望の密度でシードマクロファージ。このプロトコルでは、ウェル3.0×10 5細胞の密度/を使用した。 </li><li>感染の所望多重度（MOI）で対数増殖期における真菌細胞を追加します。このプロトコルは、MOI（ - 5.0 0.2）の範囲を収容することができます。 H.とRAW264.7マクロファージの感染カプスラーツム 、私たちは、MOI 0.2を使用していました。 </li><li>食作用を可能にするために1.5時間後に、細胞外の菌を除去するためにPBSでマクロファージを三回洗浄する。 </li><li>試料分析の前に、所望の数の時間インキュベートする。 （我々の実験では0.2）、低MOIで感染したマクロファージ細胞は数日間生存したままで、そしてサンプルは、およそ12〜24時間を分析することができる。 </li></ol> 2。マクロファージ溶解<ol><li>マクロファージ細胞から真菌を解放するには、メディアを取り出し、PBSで3回洗浄し、0.5ミリリットル滅菌水を追加します。これらの条件下では、マクロファージが溶解され、真菌細胞は、そのまま残ります。 </li><li>室温で5分間インキュベートする。 </li><li>滅菌チューブにライセートを移し、氷上に保つ。 </li><li>別のチューブに20μlのライセートを移し、その後20μlのAO / PIソリューションを追加します。ステップ5に直接進んでください： &quot;画像サイトメトリー分析のためのサンプルの準備&quot; </li></ol> 3。 CFUメッキ<ol><li>メディアにおける溶解物の10倍希釈（ステップ2.3から）実行します。 </li><li> HMM-アガロースプレート上重複の各希釈のプレートを100μl、、。 7-8日、5％CO 2で37℃で加湿チャンバー内のプレートをインキュベートする。 </li><li>手動で100の最小値と1,000異なるコロニーの最大値を表示するプレート上のコロニーを数える。 </li></ol> 4。ライブマクロファージ内の菌の可視化<ol><li>ライブマクロファージを収集するには、PBSで細胞を3回洗浄する。 4℃で30分、0.5 mlのPBSとインキュベートを追加</li><li>組織培養ウェルからマクロファージを削除するには、軽く数回上下にピペッティングし。滅菌チューブに液体を移し、氷上に保つ。 </li><li>別のチューブに20μlのサンプルを転送し、次に20μlのAO / PIソリューションを追加します。 &quot;画像サイトメトリー分析のためのサンプルの準備&quot;：ステップ5に直接進んでください。 </li></ol> 5。画像サイトメトリー分析のためのサンプル調製<ol><li>徹底的にターゲットサンプルをピペットで、その後使い捨て細胞計数室にサンプルを20μlを移す。 </li><li>細胞は、30秒のためにチャンバー内に定住することができます。 </li><li>画像サイトメーターにカウントチャンバーを挿入します。 </li></ol> 6。 Cellometer機器設定<ol><li>システムにVB-535から402とVB-660から502まで、それらが所定の位置にロックされていることを確認してください：蛍光光学モジュールを挿入します。 <ol><li> VB-535から402（475 nmで励起、535 nmでの発光）がアクリジンオレンジおよびGFPの検出に用いられる。 </li><li> VB-660から502（excitati上で540nmで、660nmの放射）を、ヨウ化プロピジウムの検出に用いられる。 </li></ol></li><li>画像サイトメーターの電源を入れ、付属のソフトウェアを開きます。 </li></ol> 7。 Cellometerソフトウェアのセットアップ<ol><li>プリセット &quot;アッセイタイプ&quot;とアッセイドロップダウンメニューで &quot;セルの種類&quot;を選択します。 <ol><li>生存率については、アッセイは、アクリジンオレンジおよびヨウ化プロピジウム検出のために最適化されている。 </li><li> カンジダ·アルビカンス感染の検出のために、アッセイはGFP検出のために最適化される。 </li></ol></li><li>上部の &quot;オプション&quot;を選択し、 &quot;を取る背景画像&quot;をクリックし、操作を完了することができます。 </li><li> &quot;プレビュー明視野像&quot;をクリックしてください。 </li></ol> 8。画像の取得手順<ol><li>画像サイトメーターに準備試料室に挿入します。 </li><li>フォーカスノブを使用して、フォーカスを調整します。 </li><li>一度ピントで、 &quot;カウント&quot;をクリックし、画像取得操作を完了することができます。 </li><li>レモ使い捨てカウンティングチャンバーをもらって適切に処分する。 </li></ol> 9。画像データ解析<ol><li>濃度及び生存率測定<ol><li>カウントが完了すると、標的細胞の濃度及び生存率を結果ページに表示される。 </li><li> カンジダ·アルビカンス感染実験におけるGFPの細胞集団の解析のためにFCSエクスプレス4にデータをエクスポートするには、 &quot;エクスポート&quot;をクリックしてください。 </li></ol></li><li> カンジダ感染細胞のFCSエクスプレス分析<ol><li> FCSエクスプレス4へ &quot;。NXDAT&quot;ファイルをインポートし、蛍光ヒストグラムの結果をプロットします。 </li><li> カンジダ·アルビカンス感染細胞の集団のパーセンテージを決定するためにヒストグラムに細胞集団ゲートを適用する。 </li></ol></li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+susceptibility+testing+of+Candida+albicans+by+flow+cytometry.">Rapid susceptibility testing of Candida albicans by flow cytometry.</a> Journal of Clinical Microbiology. 35, 358-363 (1997).</li><li>Chan, L. L. -. Y., Lai, N., Wang, E., Smith, T., Yang, X., Lin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+detection+method+for+apoptosis+and+necrosis+measurement+using+the+Cellometer+imaging+cytometry.">A rapid detection method for apoptosis and necrosis measurement using the Cellometer imaging cytometry.</a> Apoptosis. 16, 1295-1303 (2011).</li><li>Chan, L. L. -. Y., Shen, D., Wilkinson, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mating+type-like+locus+in+the+asexual+pathogenic+yeast+Candida+albicans.">Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.</a> Science. 285, 1271-1275 (1999).</li><li>Berkes, C. A., Chan, L. L., Wilkinson, A., Paradis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+of+Pathogenic+Fungi+by+Cellometer+Image-Based+Cytometry.">Rapid Quantification of Pathogenic Fungi by Cellometer Image-Based Cytometry.</a> J. Micro. Meth. 91, 468-476 (2012).</li><li>Nguyen, V. 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作家レオ李英ちゃんとベンジャミンパラディはNexcelomバイオサイエンスLLCの従業員です。

イメージサイトメトリーにより、ユーザは高品質の画像をキャプチャし、特殊なソフトウェアを使用して、細胞の迅速な定量化を行うことができる。病原微生物学分野における画像メトリーの適用つの潜在的課題は、カウントされる微生物は、哺乳動物宿主細胞を含む細胞の混合集団中に存在することである。ここでは、イメージサイトメトリーをin vitroマクロファージ感染中?...

彼らのホストおよび/または宿主細胞に伴う病原性真菌の研究は、多くの場合、時間経過を介して、または別の感染条件下で実行可能な真菌細胞の定量化を必要とする。コロニー形成単位（CFU）の列挙は、生菌数を測定したが、この手法はいくつかの欠点と限界を有することによって標準的な方法である。まず、多くの真菌種は成長が遅いです。固体培地上に見えるコロニーの成長が著しく、研究のペースを遅くする、1〜2週間かかる場合があります。いくつかの希釈がコロニーの可算数を確保するためにメッキしなければならないので、第二に、CFUメッキ中にサンプルの操作は、骨の折れるプロセスです。メッキ効率がよく、100％を下回っているので、第三に、CFU数が蒔い生菌の数よりも一般的に低くなっています。例えば、二形真菌病原体ヒストプラスマカプするめっき効率は90％と高いよいが、日常的な限り低くすることができ30％とは、関連する真菌dimorphのParacoccidioidesのパラゴムノキ 1、2のために（10％）であっても低くなっています。 カンジダ用めっき効率も変動3の対象となります。最後に、固体培地上で成長を確立することができる唯一の​​ライブと活発に分裂する細胞のためのCFU分析アカウントは、多くの状況で、一方、それは死んで、および/または代謝的に不活性な細胞の存在と濃度を決定することは有用であろう。 以前は、いくつかの病原性真菌種の定量化のためのフローサイトメトリーの方法は4-6に記載されている。ただし、共有フローサイトメーター上のバイオセーフティレベル2（BSL2）またはBSL3レベルの病原体の使用に関わるバイオセーフティ封じ込めの問題のために、この手法の採用が限定されている。フローサイトメトリーのように、イメージサイトメトリーは、細胞の定量化の感度かつ迅速な方法である。しかし、画像サイトメトリーを同等の結果7と数分の一のコストで行うことができます-11。ここでは、宿主細胞に伴って病原性真菌のイメージサイトメトリーを実行するための方法が記載されている。我々は2つの人間の真菌病原体を使用して、私たちの方法を実証： ヒストプラスマカプとカンジダをH.。カプは、呼吸器疾患を引き起こす二形真菌病原体であり、ヒトでは、出芽酵母として成長し、肺胞マクロファージ内で複製カンジダは時折カンジダ症の原因となる人間の共生種である。我々は、画像メトリが可視化機能と一緒に、これらの酵母の迅速な定量化を可能にすることを示している。

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use of image cytometry for quantification of pathogenic fungi in association with host cells

例えば、 カンジダ·アルビカンス 、 ヒストプラスマカプ 、およびクリプトコッカス·ネオフォルマンスなどの病原性酵母の細胞病原性機構の研究は、一般に、単位分析またはフローサイトメトリーを形成するコロニーを用いて酵母定量化に続いて、哺乳動物宿主又は宿主細胞（ すなわち、マクロファージ）の感染を用いる。コロニー形成単位の列挙は、フィールドで最も一般的に使用される方法であったが、この技術は成長を比較するときに特に懸念される固体培地および低および/または可変メッキ効率、上のいくつかの真菌種のゆっくりとした成長を含めて、不利な点と制限があります野生型および変異株である。フローサイトメトリーは、酵母の生存率に関する迅速な定量的情報を提供することができるが、病原性酵母のフローサイトメトリー検出の適用は、その高コスト及び生物学的安全性の考慮を含む実用的な理由の数が限られている。ここでは、イメージベースのデモンストレーションマクロファージとの共培養中の生存病原性酵母の定量化のためのCellometerビジョン（バイオサイエンスNexcelom、LLC）を使用してフローサイトメトリーの方法論。 2人間の真菌病原体の検出に関する我々の研究の焦点： ヒストプラスマカプとカンジダH.。カプはマクロファージファゴソーム内で複製することによって肺胞マクロファージコロニーを形成し、ここで、我々は定量H.の成長を評価するイメージサイトメトリーと組み合わせて、アクリジンオレンジ/ヨウ化プロピジウム染色を用いてRAW 264.7マクロファージにおけるカプ酵母。我々の忠実方法従来のコロニー形成単位の列挙によって測定された反復するの成長傾向が、大幅に増加した感度。さらに、我々は直接Cの GFP発現株でライブマクロファージの感染性を評価アルビカンス 。我々の方法論は、アソシエーションのウィットに重要なヒトの真菌病原体の検出および定量のために、迅速、正確、かつ経済的な手段を提供しています時間宿主細胞。

我々は、H.の成長を監視するCellometerビジョン画像サイトメーターを用いマクロファージ細胞内カプスラーツム 。 RAW 264.7細胞はH.に感染していたカプ酵母細胞及び様々な時点で、サンプルは画像ベースのサイトメトリー解析に続いAO / PI染色を行った。並行して、サンプルは、従来のCFU列挙によって分析した。各時点において、サンプルは、マクロファージを溶解す?...

Watch this Scientific Journal Video about Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells at JoVE.com

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

ここでは、イメージサイトメトリー、培養中の宿主細胞に対応付けて病原性真菌の定量に使用することができる方法を示す。この技術は、CFU列挙の代替として使用することができる。

宿主細胞に関連して病原性真菌の定量化のための画像サイトメトリーの使用

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

use-image-cytometry-for-quantification-pathogenic-fungi-association

Research

JoVE Journal

Immunology and Infection

12.8K ビュー.  Merrimack College. ここでは、イメージサイトメトリー、培養中の宿主細胞に対応付けて病原性真菌の定量に使用することができる方法を示す。この技術は、CFU列挙の代替として使用することができる。

ビデオ: Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

フローサイトメトリーによって負傷した脊髄組織における免疫細胞の定量的評価：セルの精製方法のための新しい使用法

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

免疫・感染学

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

動的な生細胞イメージングのための宿主 - 病原体相互作用の研究のための光トラップの使用

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

長期宿主 - 病原体相互作用を特徴付けるためのEpiAirwayモデルの利用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

哺乳動物細胞に関連して細菌の生存を決定するための蛍光顕微鏡法

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

宿主細胞と嫌気性菌の相互作用を評価するためのモデル生物としてポルフィロモナス・ジンジバリス

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

T細胞におけるクロマチンの状態を評価するためにフローサイトメトリーの使用

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

ミトコンドリア、リソソーム T 細胞のマルチカラー フロー フローサイトメトリーを用いた定量化

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

2型糖尿病と関連した非アルコール性脂肪性肝炎のための高度なマウスモデル

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

ヒト骨髄性U937細胞におけるフローサイトメトリーによるSAMHD1制限の解析

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

結核の宿主向け治療の前臨床試験に使用する自動培養システム

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...