保健研究、カナダの白血病とリンパ腫協会とカナダのリンパ腫財団のカナダの協会からの補助金によって支え。

試薬を準備する注：すべてのPBSは、Ca 2 +およびMg 2を含む+ <ol><li>検出試薬 室温のPBSに20μM7 - アミノ-4 - クロロメチルクマリン、T - BOC -ロイシン - メチオニンアミド（BOC - LM - CMAC）を追加します。各細胞懸濁液は、2mLの検出試薬が必要になります。 </li><li> 1％パラホルムアルデヒド（固定セル） 室温のPBS中4％パラホルムアルデヒドのストック溶液を希釈する。各FACSチューブへ分注し1mLの1％パラホルムアルデヒド。三FACSチューブは、各実験条件（例：32Dkit、32Dkit + PD150606、32Dkit + PD98059必要性9 FACSチューブ）のために必要です。 </li><li> PD150606（カルパイン阻害剤） 100mmの最終濃度にDMSOで再構成PD150606。この試薬と、この実験の期間中、軽いからそれで処理するすべてのセルを保護する。 </li><li> PD98059（MEK1阻害剤） 50mmの最終濃度にDMSOで再構成PD98059。 </li></ol>セルを準備する<ol><li> IL - 3（またはIL - 3などのための他のソースのソースとして5％ウシ胎児血清、WEHI - 3上清を添加したのOpti - MEM培地中でmL当たり5 × 10 5〜1 × 10 6細胞で32Dkit細胞を成長させる組換えタンパク質として）、0.1％2 - メルカプトエタノール及び抗生物質。 </li><li> three 15mlのファルコンチューブ（試験管当たり4 × 10 6細胞）に12 × 10 6 32Dkit細胞を集めます。 </li><li> 15℃5分間1000rpmでペレットは、細胞を℃に</li><li>上清を吸引除去する。 2mLの室温のPBSで細胞を再懸濁します。 </li><li> 50μMPD150606で一つの細胞懸濁液を扱います。サンプルをアルミ箔でファルコンチューブを被覆して光から保護されていることを確認します。ボルテックスは簡潔にして暗所で室温で20分から30分インキュベートする。 </li><li> 20μMPD98059と第二の細胞懸濁液を扱います。板37で2時間35ミリメートル組織培養皿とインキュベートにおける細胞℃に</li></ol>固定化細胞におけるカルパインのアッセイ<ol><li>サンプルはPD150606と共にインキュベートされているが、未処理の試料でカルパインアッセイを開始します。各細胞懸濁液に2mLの検出試薬を追加することから始めます。直ちに1％パラホルムアルデヒド（0分の時点）で事前に準備されたFACSチューブに細胞懸濁液/検出試薬の混合液1mLを加える。 </li><li>室温で5分間検出試薬で細胞懸濁液をインキュベートする。次に1mLの1％パラホルムアルデヒドでFACSチューブに細胞懸濁液1mLを加える。 </li><li>室温でさらに5分間検出混合物を用いて細胞懸濁液をインキュベート続ける。次に1mLの1％パラホルムアルデヒドでFACSチューブに細胞懸濁液1mLを加える。 </li><li>阻害剤で治療されている32Dkit細胞のカルパインのアッセイを繰り返します。 </li><li> 4℃で暗所で一晩細胞を固定℃に</li><li>翌日ペレット15℃、5分間1800rpmで細胞を℃に</li><li>上清を吸引除去する。 750uL PBSで細胞を再懸濁します。氷の上に細胞を配置し、暗所で保管。 </li></ol>固定セルのデータを取得する<ol><li> LSR II（BDバイオサイエンス）を用いて細胞を分析する。フィルタは、光Xcyte（UV）レーザー、励起線波長355nm、レーザーパワー25mWを、それぞれ基板と製品の405と450nmの発光のために設定されています。基質と生成物のためのバンドパスフィルタは、20分の405と50分の450です。基質と生成物のためのロングパスフィルターは、405と450です。 PMT電圧は、製品の基板のための350から375と325から350に設定されました。 </li><li> 、側方散乱光、インド - 1ブルー（ログ）、インド- 1バイオレット（ログ）前方散乱：次のパラメータを持つBD FACSDiva実験アップを設定します。前方散乱対生きた細胞でのゲート（図1a）、インド- 1ブルーヒストグラム、インド- 1バイオレットのヒストグラム、インド - 1ブルー対インド- 1バイオレットと側方散乱ドットプロット：ワークシートに以下のグラフを設定するドットプロット。 </li><li> AlignFlowとAlignFlowプラス蛍光ビーズ（Invitrogen）でLSR IIを校正します。 PMT電圧は前の実験ごとに同じチャンネルでビーズ蛍光ヒストグラムのピークを配置するために調整する必要があります。 </li><li> 0分の時点で32Dkitセルを使用してデータの取得を開始。必要に応じて、パラメータの電圧を調整します。サンプル10,000個のイベントを取得し、記録する。 </li><li>各チューブの間にFACSバッファーでマシンをすすぎ、各サンプルのために買収を繰り返します。 </li><li>データをエクスポートします。 LSR IIは、研究所のガイドラインにしたがって洗浄されていることを確認します。 </li></ol>生細胞におけるカルパインのアッセイ<ol><li>ないPD150606と、上記のように細胞懸濁液を準備する。 LSR IIにサンプルを持参し、上記のようにフィルタとパラメータを設定します。上記のようにワークシートを設定します。インド- 1ブルー対時間を示す大規模なドットプロットが含まれています。最大numを取得するプログラムを設定するイベントのBER（無停止のゲート）。 </li><li> 32Dkit細胞の50μMPD150606 1つの細胞懸濁液を追加します。暗闇の中でサンプルを保ち、20〜30分室温でインキュベートする。 </li><li>ないPD150606と32D​​kit細胞懸濁液上にデータの取得を開始。毎秒200から300までのイベントの低流量を使用してください。 30秒から1分後にマシンからチューブを取り外します。細胞に20μMBOC - LM - CMACを追加。ボルテックスは簡潔にしてデータを取得し続けます。 </li><li> 10-20分間、またはカルパイン活性がプラトーになるまでデータを取得する。 </li><li> PD150606で処理32Dkit細胞と、手順3と4を繰り返します。 </li><li>データをエクスポートします。 LSR IIは、研究所のガイドラインにしたがって洗浄されていることを確認します。 </li></ol>コンプレックス（混合）細胞集団におけるカルパインのアッセイ *この実験は、マウスの脾臓から採取した細胞懸濁液に32Dkit細胞を識別します。 <ol><li>各マウスから脾臓を回収する。室温で10分間、NH 4 Clで赤血球を溶解する。 </li><li> 2mLのPBSで単一の細胞懸濁液を準備します。 </li><li>二つに各細胞懸濁液を分ける。暗所で室温で20〜30分間、50μMPD150606 1細胞懸濁液を扱います。 </li><li>固定化細胞におけるカルパインアッセイ（ステップ1-6）のために上記のように進んでください</li><li>上清を吸引除去する。 500μLのPBSで細胞を洗浄。上清を吸引除去する。 </li><li> 2％BSAを200μL染色バッファーで細胞を再懸濁します。室温で15分間インキュベートする。 </li><li>一次抗体（FITC抗マウスCD117）を準備する。抗体は、バッファを染色で1:200希釈で使用してください。抗体は暗所で保管されていることを確認します。 </li><li>細胞に200μL一次抗体を追加。暗所で室温で30分間インキュベートする。 </li><li>フローサイトメトリー用のボリュームを増加させる400μLPBSを追加。氷の上に細胞を配置し、暗所で保管。 </li><li>固定セルのデータを取得で説明されている手順に進みます。パラメータにFITC追加し、ワークシート上にFITCのヒストグラムが含まれています。 </li></ol>分析と代表の結果<ol><li> FlowJo（Treestar）を使用してデータを分析する。前方および側方散乱プロファイル（図1a）に基づいて、生細胞でのゲート。生細胞のゲート（図1b）からインドブルーの平均蛍光を決定します。 </li><li>混合細胞集団の場合） <ul><li> 32Dkit人口を識別するために、ヒストグラム、ドットプロットにFITCの蛍光を使用してください</li><li> 32Dkitの人口のインド - ブルーの平均蛍光を決定する</li></ul></li><li>平均蛍光（図2a）は、グラフにMicrosoft Excelを使用してください。 5〜10分の間の直線の傾きを計算する。グラフの傾き（図2b）細胞におけるカルパインの活性を決定するために棒グラフとして。 </li></ol><img src="/files/ftp_upload/2050/2050fig1a.jpg" alt="図1a" /> 図1a。前方および側方散乱プロファイルに基づいて生細胞を決定する。 <img src="/files/ftp_upload/2050/2050fig1b.jpg" alt="図1b" /> 図1bは、10分の時間経過とともに蛍光変化を平均。 <img src="/files/ftp_upload/2050/2050fig2a.jpg" alt="図2a" /> 図2a。平均蛍光の代表グラフ。 <img src="/files/ftp_upload/2050/2050fig2b.jpg" alt="図2b" /> インヒビターを有する32Dkit細胞における図2b。カルパイン活性。

<ol>
	<li>Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Calpain System. Physiol. Rev. 83, 731-801 (2003).</li><li>Niapour, M., Berger, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometric+Measurement+of+Calpain+Activity+in+Living+Cells.">Flow Cytometric Measurement of Calpain Activity in Living Cells.</a> Cytometry A. 71A, 475-485 (2007).</li><li>Leloup, L., Daury, L., Maz&#233;res, G., Cottin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+ERK/MAP+kinase+signalling+pathway+in+milli-calpain+activation+and+myogenic+cell+migration.">Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration.</a> The International Journal of Biochemistry and Cell Biology. 39, 1177-1189 (2007).</li></ol>

利害の衝突は宣言されません。

固定と生活32Dkit細胞におけるカルパイン活性はこのビデオのプロトコルで決定されている。カルパイン阻害剤PD150606を有する細胞系の治療は、細胞内のカルパイン活性の完全な阻害をもたらした。さらに、MEK1阻害剤PD98059による治療は、細胞内のカルパイン活性の完全な阻害をもたらした。 ERKは、カルパイン- 2 3の主要な活性剤です。これらの結果は、カルパイン- 2活性が32Dkit細胞で?...

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<td>PBS BOC-LM-CMAC 4% paraformaldehyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td> Dissolve 4g paraformaldehyde in 100mL PBS. In fume hood, heat sample with stirring until clear. Let cool then store at 4&deg;C</td>
</tr>
<tr>
<td>PD150606</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>Use freshly reconstituted PD150606 for maximal calpain inhibition.</td>
</tr>
<tr>
<td>PD98059</td>
<td>&nbsp;</td>
<td>Cell Signaling Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>AlignFlow, AlignFlow plus fluorescent beads FACS tubes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>LSR II</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>&nbsp;</td>
<td> Supernatant of WEHI-3 cells is used as a source of IL-3 in these experiments. Each collection of WEHI-3 supernatant is titrated to determine the optimal concentration used for 32Dkit cell growth. </td>
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measuring calpain activity in fixed and living cells by flow cytometry

カルパインは、ユビキタス細胞、カルシウム感受性、中立的なシステインプロテアーゼです。<sup&gt; 1</sup&gt;。カルパインは、シグナル伝達、細胞骨格のリモデリング、遺伝子発現の調節、アポトーシスや細胞周期の進行を含め、多くの生理学的プロセスにおいて重要な役割を果たす<sup&gt; 1</sup&gt;。カルパインは、筋ジストロフィー、癌、糖尿病、アルツハイマー病や多発性硬化症を含む多くの病理に関与している<sup&gt; 1</sup&gt;。カルパイン規制は複雑で、完全には理解です。 mRNAおよびタンパク質レベルでは、カルパイン活性を測定するための遺伝子またはタンパク質の発現技術の使用を制限し、活動にあまり相関しない。このビデオのプロトコルの詳細については、固定および生細胞中のカルパイン活性を測定するための我々の研究室では、フローサイトメトリーアッセイを開発した。このメソッドは、カルパインにより特異的に切断される蛍光基質BOC - LM - CMACは、、カルパイン活性を測定するために使用します。<sup&gt; 2</sup&gt;このビデオでは、固定および生体マウス32Dkitの白血病細胞、単独で、または脾細胞集団の一部としてのカルパイン活性はLSRII（BDバイオサイエンス）を用いて測定されます。 32Dkit細胞は、正常脾細胞と比較して上昇活性を有することが示されている。

Watch this Scientific Journal Video about Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry at JoVE.com

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

この記事では、フローサイトメトリーを用いて固定し、生きた細胞内のカルパイン活性を測定するための詳細プロトコルを務める。

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, ...

measuring-calpain-activity-in-fixed-and-living-cells-by-flow-cytometry

Research

JoVE Journal

Immunology and Infection

11.7K ビュー.  University of Toronto. この記事では、フローサイトメトリーを用いて固定し、生きた細胞内のカルパイン活性を測定するための詳細プロトコルを務める。

ビデオ: Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

免疫・感染学

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

フローサイトメトリーによって、A549細胞におけるインフルエンザAウイルスの添付ファイルと内在化を測定

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

離とマウスとヒトの肺胞マクロファージの体外培養

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

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

好中球系統細胞の質量細胞法解析に対する全骨髄の調製

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

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 ...

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

フローサイトメトリーを用いた赤血球補体受容体1の測定

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

フローサイトメトリーベースアッセイによる熱帯 熱マラリア原 虫に対する自然獲得食細胞症誘導抗体の測定

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

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 ...