著者は、映画撮影のためのテクニックと氏ポールシェパードを実行するためのミスローラマシューズに感謝したいと思います。このプロジェクトは、南オーストラリア大学、研究室医学のプログラムによって資金を供給された。

5倍のベロナール緩衝生理食塩水の調製（VBS） <ol><li>ベロナール緩衝生理食塩水（VBS）を準備するには、3つの別々のソリューションが用意する必要があります。 </li><li>蒸留水の350ミリリットルでナトリウムBarbitoneのNaClおよび0.94gmの21.25gmを溶解して溶液1を準備します。 NaClおよびナトリウムBarbitoneの最終濃度は、それぞれ1.02Mと13mmです。 </li><li>熱い蒸留水の125​​ミリリットルでBarbitoneの1.44gmを溶解して溶液2を準備します。 Barbitoneの最終濃度が62.5mMである。 </li><li>蒸留水の100mlにCaCl 2のMgCl 2と4.41gmの20.33gmを溶解して溶液3を準備。 MgCl 2とCaCl 2の最終濃度は2.18M、それぞれ440mMである。 </li><li>ミックスのソリューション1と2と室温まで冷却。 </li><li>組み合わせた溶液を冷却した後、溶液3の1.25ミリリットルを追加し、1M HClを用いて7.3から7.5にpHを調整する。 </li><li> 5倍のストック溶液を調製するために蒸留水で500ミリリットルに、最終的な音量を調整します。 </li><li> 1Xワーキング溶液を調製するために、蒸留水で株式1:5に希釈する。 </li></ol>溶血素とヒツジ赤血球の感作性<ol><li>まず、VBSで1：50希釈して溶血を準備</li><li> VBSのSRBCを追加6ミリリットルの4ミリリットルに、穏やかに転倒混和</li><li> 600グラムX 5分間遠心</li><li>上清を捨て、細胞をさらに2回洗浄</li><li>最終洗浄後、細胞をパックに900グラムのx 5分で細胞を遠心分離</li><li>上清を破棄して、パックされた細胞の10％溶液、すなわち0.5ミリリットルを準備するのに十分なVBSで細胞を再懸は、緩衝液5 mlに再懸濁し、 </li><li>継続的に渦巻くながら、細胞への溶血素の等量（ウサギ抗ヒツジ赤血球抗体）を追加滴下</li><li>水浴で30分間30℃でインキュベートする</li><li>静かに細胞ごとに15分を混ぜて</li><li> Sensitised SRBCは4℃で一晩保存することができます</li></ol> CH 50アッセイ<ol><li>午前1時08分、1:16、1:32、1:64と1:128との重複の管の一連のラベルを付けます。 </li><li>重複してVBSで制御し、試験血清の2倍連続希釈それぞれのシリーズを準備</li><li> 1:4（100ミリリットル血清+ 300ミリリットルVBS）で起動し、次のラベル付きチューブにサンプルの200ミリリットルを転送する。 </li><li>新鮮なピペットの先端で、次の希釈に希釈し、移転の200ミリリットルの間で充分に混合する。 </li><li>すべての5つの希釈が行われるまで繰り返します。 </li><li>最後の1:128希釈から200ミリリットルを捨てる。 </li><li>すべてのチューブに懸濁感SRBCの200ミリリットルを追加。 </li><li>ラベルつの異なるチューブは、BLANKと感作SRBC + 200ミリリットルVBSの200ミリリットルを追加します。これらのチューブは、VBSでSRBCの自発的な溶解を測定します。 </li><li> 総溶解などの別の2つの別々のチューブにラベルを付け、感作SRBC + 200ミリリットルの蒸留水200ミリリットルを追加します。 </li><li>静かにすべてのチューブを混ぜる。 </li><li> 37℃、15分後に混合水浴で30分間インキュベートします。 </li><li>堆積物に5分間赤血球、1,500 gで、サンプルを遠心分離します。 </li><li> 96ウェル平底プレートにウェルに各チューブから上清の100ミリリットルを転送する。 </li><li>各ウェルに蒸留水の100ミリリットルを追加。 </li><li>プレート分光光度計を用いて540nmでサンプルの吸光度を読み取る。 </li></ol>計算<ol><li>各サンプルについての平均吸光度を計算する</li><li>すべてのサンプルからブランク吸光度（自発的な溶解を）引く</li><li>次の式を使用して、各希釈用％の溶解を計算します。 <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="式" /></li><li>横軸上に血清希釈に対する割合の溶解（縦軸）をプロットする。 </li><li>制御および試験血清の50％の溶血（図2）に必要な希釈を計算します。 </li></ol>代表的な結果： ビデオでは、代表的な結果の例が含まれています。実際には、コントロール血清はまた、テストの血清と同時に実行され、同じ方法で扱われます。以下の（表1）テストされた血清サンプルからサンプルデータです。データは5.3で示した方程式にしたがって操作されます。 <table border="1"><tbody><tr><td> サンプル </td><td> OD 540 </td><td> 意味 </td><td></td><td></td></tr><tr><td>空</td><td> 0.042、0.044 </td><td> 0.043 </td><td></td><td></td></tr><tr><td>総溶解</td><td> 0.183、0.183 </td><td> 0.183 </td><td></td><td></td></tr><tr><td colspan="5"></td></tr><tr><td> 希釈 </td><td> OD 540 </td><td> 意味 </td><td> 平均ブランク </td><td> ％の溶解 </td></tr><tr><td>午前1時08分</td><td> 0.200、0.219 </td><td> 0.210 </td><td> 0.168 </td><td> 116 </td></tr><tr><td> 1時16分</td><td> 0.179、0.173 </td><td> 0.176 </td><td> 0.134 </td><td> 93 </td></tr><tr><td> 1時32分</td><td> 0.134、0.110 </td><td> 0.122 </td><td> 0.08 </td><td> 55 </td></tr><tr><td> 1:64 </td><td> 0.053、0.066 </td><td> 0.059 </td><td> 0.017 </td><td> 12 </td></tr><tr><td> 1:128 </td><td> 0.044、0.045 </td><td> 0.045 </td><td> 0.003 </td><td> 2 </td></tr></tbody></table><img src="http://www.jove.com/files/ftp_upload/1923/1923fig1.jpg" alt="図1" /> 図1。古典的補体経路の活性化。古典経路は、標的細胞の表面に結合免疫グロブリンM（IgM抗体）または免疫グロブリンG（IgG）をによって活性化される。抗体のFc部分がC1qに結合し、C1Rが活性化し、これは順番に一緒にC1sの2つの分子を活性化するC1Rの別の分子を活性化される。 C1sのは、今も切断されるC2の結合部位を公開してC4を切断する。 C4BとC2Aの結合は、C3転換と呼ばれる複合体の形成を導く。この複合体は今C3成形C3aとC3bを切断、C5の転換を形成するC3転換と組み合わせるそのうちのいくつか。この複合体は、現在の膜攻撃複合体（MAC）の形成を開始するC6への結合の結果C5bと、C5に作用する。で、最終的なMACの形成をもたらすC9で、最終的にはC8に行為をオンにしてC7でC5b6行為、。 （Goldsby ら 、2003）。 <img src="http://www.jove.com/files/ftp_upload/1923/1923fig2.jpg" alt="図2" /> 図2。サンプルデータをプロットし、制御と試験血清サンプルのCH50を計算する。（）、計算された割合（％）対照（•）と試験（両方の溶解は）血清サンプルを希釈率に対してプロットされています。それはグラフの線と交差して、縦線が希薄にダウン描画されるまで、（B）50％の溶解を計算するには、ラインは50％のパーセント値から描画されます。この例では、制御および試験血清の21.6倍に希釈、35倍希釈、50％の溶解を達成するために必要である。このデータは、50％の溶解に到達される前に以下の希釈、必要に応じて、コントロールに比べてテストの血清中に存在する以下の補数があることを示します。対照サンプルには、1:8の希釈で&gt; 100％の溶解を示しています。これは、多くの場合、補体成分を蒸留水とより効率的な溶解によりSRBCの不完全な溶解によるものです。

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, 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> Immunology. , (2003).</li></ol>

CH 50アッセイは、多くの干渉の対象となります。いくつかのSRBCでの活動を補完する関係がない自発的な溶血の結果、他のものより壊れやすいものです。ウサギの抗体の親和性はロットし、1つのメーカーによって異なりますが、これはSRBCに結合する抗体の量に影響を与えます。また、抗体のコーティングの異なる量のSRBCによる細胞内で抗体の結果とSRBC感作のプロセス。検体採取と保...

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<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
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<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Human serum</td>
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<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
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<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
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<td>Veronal buffered saline</td>
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<td>&nbsp;</td>
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<td>Use at 1x final</td>
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measuring the 50% haemolytic complement (ch50) activity of serum

補体系は、細胞溶解をターゲットにつながる活性化とopsonisationを通して食作用を促進するタンパク質のグループです。個々の補体成分は、しかし、これは経路の活性などの情報を提供しない定量することができる。 CH<sub&gt; 50</sub&gt;古典的補体経路（図1）の活性化のためのスクリーニングアッセイであり、経路の任意のコンポーネントの削減、不在および/または非アクティブに敏感です。 CH<sub&gt; 50</sub&gt;ウサギ抗ヒツジ赤血球抗体（溶血素）をプレコートヒツジ赤血球（SRBC）を溶解するために古典経路の血清補体成分の機能的能力をテストします。抗体でコーティングされたSRBCは試験血清、補体古典経路が活性化され、溶血の結果とインキュベートされている場合。補体成分が存在しない場合、CH<sub&gt; 50</sub&gt;レベルはゼロになります。古典経路の1つまたは複数のコンポーネントが減少している場合は、CH<sub&gt; 50</sub&gt;縮小されます。最適に感作されたSRBCの固定量は、各血清希釈に追加されます。インキュベーション後、混合物を遠心分離し、溶血の程度は540nmで上清中に放出されたヘモグロビンの吸光度を測定することによって定量される。補体活性の量は、抗体コーティングされたSRBCを溶解するために血清検査の種々の希釈物の容量を調べることによって決定されます。このビデオでは、古典的補体経路の補体活性のレベルを分析に関わる実験的な手順を説明します。

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

古典経路は、抗体によって活性化し、標的細胞の溶解で最高潮に達しますれる。 CH<sub&gt; 50</sub&gt;アッセイは、血清サンプルの補体活性の尺度を提供する。このビデオでは、CHを決定する際に必要な手順を示しています<sub&gt; 50</sub&gt;血清サンプルの、結果の計算と解釈する。

50パーセント溶血補体（CHの測定 50</sub血清中>）活動

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

37.4K ビュー.  University of South Australia. 古典経路は、抗体によって活性化し、標的細胞の溶解で最高潮に達しますれる。 CH 50アッセイは、血清サンプルの補体活性の尺度を提供する。このビデオでは、CHを決定する際に必要な手順を示しています 50血清サンプルの、結果の計算と解釈する。

ビデオ: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

補体の枯渇により特定の細胞集団の枯渇

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

免疫・感染学

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

赤血球凝集アッセイを用いて血清の反応性と力価の決定

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

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

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

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

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

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

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

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

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

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

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

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

臨床抗体サンプルの貪食活性を決定する

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

赤血球上の抗体誘導性補体活性化の定量的検出のための方法

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

マウスにおけるローカルアナフィラキシーの測定

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

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

ヒト血液中の測定顆粒球と単球食作用および酸化バースト活動

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

細菌の付着と血清抵抗性ビトロネクチンの役割を研究するための試金

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

50パーセント溶血補体（CHの測定 50）活動

50パーセント溶血補体（CHの測定_50）活動