私は図1のデータを提供するためSreemanti Basuさんに感謝します。この作品は、NIHの助成金AI069358とBloodCenter研究財団によって部分的にサポートされていました。

A.ウサギの補数<ol><li>株式のウサギの補数は、室温で解凍し、0.5および/または1ミリリットルボリュームに等分し、℃で長期保存のための-20-80で凍結保存してください。 </li><li>補体の各ロットは、最適な作業濃度を決定するために滴定する必要があります。一般的に、1:10-1:20の希釈が最適です。 </li><li>ウサギ補体は常に細胞の最終用途に応じて、販売前に滅菌済みされていない分注し、凍結または細胞に添加する前にするとき、補体を滅菌したフィルタリングする必要があります。 </li></ol> B.抗体の選択<ol><li>すべての抗体はこのように抗体が最初に活性についてテストされる必要があります、補体活性で有効である。 </li><li>マウスとラットモノクローナル抗体の両方は、補体活性化に効果的です。 </li><li> IgM抗体はIgGに続く補体による細胞溶解に使用するため、一般的に最も効率的です。最高の補体活性化のためのIgGアイソタイプは、種によって異なります。 </li><li>各抗体は、10 7細胞/ mlの細胞密度での最適な使用のために滴定する必要があります。 </li></ol> C.基本的なプロトコル<ol><li>熱不活性化FCSを含む培地中で× 10 7細胞/ ml 5に開始する細胞集団を調整します。細胞の最終容量は倍の起​​動ボリュームになります。枯渇が定量化できるように、いくつかのセルを保存することを忘れないでください。 </li><li>各抗体は、適切な所定の濃度または希釈で細胞懸濁液に追加する必要があります。よく反転により細胞を混ぜる。 </li><li>必要な補体の量を計算します。室温で補数の株式を解凍。補数が滅菌されていない場合、それは低タンパク結合シリンジチップフィルターを使用して滅菌する。注射器に、補体を添加する前に培地のいくつかのmlを追加し、細胞と抗体を含むチューブに直接フィルタを適用。 </li><li>細胞が1 × 10 7細胞/ mlになるようにチューブの最終容量を調整します。 </li><li> 15分ごとに混合、一時間、37℃で細胞をインキュベートする。 </li><li>細胞を遠心し上清を捨てる。 </li><li>培地で2回洗浄してください。細胞残渣は、ポリスチレンチューブの側に残ることでしょう。死んだ細胞や残骸はセルストレーナーまたは密度勾配遠心分離によってこれを除去することができます。 </li><li>枯渇する前と後の利益の細胞集団を比較することにより、細胞の純度を確認してください。フローサイトメトリーおよび免疫組織化学、細胞の純度を決定する二つの良い方法です。 </li></ol>代表的な結果補体の活性化において非常に効率的なモノクローナル抗体を使用する場合は、1回のラウンド後の細胞の枯渇は、95％以上です。これは、我々はフローサイトメトリーによるTCRβの発現（eBioscience社、サンディエゴ、CA）によって検出されたとして、32.2％αβT細胞から成るマウス脾細胞の使用を開始図1に示します。我々は、mAb THY1のための特定の、すべてのT細胞により発現されたタンパク質が、他の白血球を用いてT細胞を枯渇。我々が使用される抗体はY - 19ラットIgG2c 2であった。記載された手順に続いて、TCRβ人口は1.56％、95％以上の削減に減少した。 <img src="/files/ftp_upload/1487/1487fig1.jpg" alt="図1" /> 図1。補体活性化によるTHY1 +脾臓T細胞の枯渇。合計マウス脾細胞は、赤血球の脾臓と溶解の均質化により得られた。アンチTHY1し、所定の濃度で赤ちゃんウサギ補体は、最終細胞濃度が1 × 10 7細胞/ mlとなるように脾細胞の単細胞懸濁液に添加した。 37℃で1時間Cのためのインキュベーション後、細胞を2回洗浄し、細胞の生存率をトリパンブルー排除により評価。枯渇の効率は、TCRβ+枯渇する前にセル（左パネル）と枯渇後（右パネル）の割合を比較するフローサイトメトリーによって評価した。水平バーは、陽性染色を示し、バー上の数字は、正のパーセントです。

<ol>
	<li>Janeway, C., Travers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immune+System+in+Health+and+Disease.">The Immune System in Health and Disease.</a> Immunobiology. , (1994).</li><li>Jones, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activities+of+antibodies+against+brain-associated+T+cell+antigens+II.+Stimulation+of+T+cell-induced+B+cell+proliferation.">Functional activities of antibodies against brain-associated T cell antigens II. Stimulation of T cell-induced B cell proliferation.</a> Eur J Immunol. 12, 30-37 (1982).</li></ol>

細胞集団の精製は、その機能の研究に必要な一般的な手順です。ここでは、抗原特異的抗体と補体の枯渇を使用して細胞集団を枯渇の迅速かつ効果的な方法を説明します。補体活性化抗体は、系統特異的なマーカーに利用できれば、どのような細胞集団の枯渇を達成することができます。ここでは、THY1のためのモノクローナル抗体特異的、本質的にすべてのT細胞集団で発現させたタンパク質を用いてT細胞の枯渇を示した。我々は、αβT細胞の95％以上が枯渇し、残りの脾細胞は、68％から98％に増加した。細胞の純度のこのレベルは、FACSおよび磁気ビーズ分離を含む他の精製戦略と似ています。補体の枯渇は、出発細胞集団が得られれば1.5時間で実行できる高速な手法です。補数の枯渇の別の利点は、高価な装置や試薬を必要としないための検査では、テクニックを実行できることです。必要な機器の唯一の主要な部分は、水浴及び遠心分離機です。ウサギの補数は、妥当なコストで、さまざまなベンダーから購入することができます（試薬の表を参照）、多くのmAbをATTCから購入し、コストを制御するために局所的に成長させることができる。対照的に、FACSは6図と高価な蛍光標識モノクローナル抗体で費用がかかります細胞選別能力を持つフローサイトメーターを必要とします。磁気ビーズの分離は、手頃な価格ですが、コストがかかります磁石と磁性ビーズ、購入する必要があります。さらに、いくつかの磁気ビーズ分離戦略はまた、カラムの購入が必要です。 細胞枯渇のための補数を使用することに主な障害とは、必要な抗原特異性と抗体の活性化補体の可用性です。ヒト、マウスやラットの抗体はすべての補数を修正する能力を持っていますが、いくつかのアイソタイプは、他よりも優れています。すべての3つの種からのIgMは補体を固定で非常に効果的であるが、IgGアイソタイプは変わる。したがって、すべてのモノクローナル抗体は、最初にその有効性を判断するためのパイロット試験で試験されている必要があります。非常に効率的ではない抗体の場合は、補体の枯渇の2ラウンドは、残りの細胞集団の生存能力に影響を与えずに行うことができます。コスト削減として、抗体は、最適な有効性のために滴定する必要があります。さらに、使用される抗体は、精製する必要はありません。ハイブリドーマ上清および腹水の作業と同様に効果的に、精製が困難なIgM抗体の場合に特に便利です。抗体と同様に、補体がパイロットスタディで調べられることが不可欠です。濃度が高すぎるが、非標的細胞集団の生存能力を失うことになります。また、重要なインキュベーション時間は60分を超えていないことです。 我々のプロトコルでは、我々は一緒に抗体や補体を共同でインキュベートする。しかし、細胞は15-30分のための抗体で標識し、前の補数の加算に洗浄することができます。この戦略を使用する場合は、のようなパッチおよびキャッピングまたは受容体の内部でいくつかのタンパク質で発生、細胞表面上の細胞の抗原の消失を防ぐために、氷上で抗体のインキュベーションを実施してください。非常に効率的ですが、他の方法に比べて枯渇を補完するための主な欠点は、正の選択のために使用することができないということです。したがって、細胞の亜集団を得ることが困難である。例えば、それは最高のFACSによって行われる濾胞性B細胞、から辺縁帯B細胞を精製するのは困難です。このような状況では、補体活性化は、ソートされているセルの数を減らすためにT細胞を枯渇させるために使用することができる。この戦略は、ソート時間を減らすことがお金の節約になりますこれと細胞の少ない厳しいだろう。 特定の細胞集団の精製は、一般的な要件であるため、多くの戦略が開発されている。補数の枯渇の戦略の主な利点は、その手頃な価格、技術的なシンプルさと時間の節約です。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>3-4 week baby rabbit complement</td>
<td>&nbsp;</td>
<td>Pel-Freez Biologicals</td>
<td>31061-3</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>&nbsp;</td>
<td>various</td>
<td>various </td>
<td>An incubator set at 37 &deg;C can also be used.</td>
</tr>		</tbody>
		</table>
		</div>

depletion of specific cell populations by complement depletion

免疫細胞集団の精製は、しばしば彼らのユニークな機能を研究するために必要です。特に、このようなリアルタイムPCRやマイクロアレイ解析などの分子生物学的なアプローチは、高純度で細胞集団の分離が必要です。一般的に使用される精製戦略は、蛍光活性化細胞選別（FACS）、磁気ビーズ分離および補体枯渇などがあります。 3つの戦略の、補数の枯渇は、高速で安価な、細胞と高い細胞収率で穏やかであるという利点を提供しています。補体系が活性化されると細胞死をもたらす細胞表面に細孔を形成する膜攻撃複合体の形成で最高潮に達するタンパク質分解カスケードを開始する血漿蛋白質の多数で構成されています<sup&gt; 1</sup&gt;。古典経路は、IgMおよびIgG抗体により活性化され、最初の細菌を殺しためのメカニズムとして記述されていた。モノクローナル抗体（mAb）の生成と、補体カスケードは、抗原特異的に任意の細胞集団を溶解するために使用することができます。補体カスケードによる細胞の枯渇は、出発細胞集団への抗原特異的抗体とウサギ補体を固定する補体の添加によって達成される。細胞は37℃で1時間インキュベート℃及び溶解した細胞は、その後、洗浄の2つのラウンドで削除されます。補体結合のための高効率でモノクローナル抗体は、通常、標的細胞集団の95〜100％を使い果たす。標的細胞集団の精製方法に応じて、補体の枯渇は細胞の精製のためか、さらにその後の方法により精製することができる細胞集団を濃縮するのに使用することができます。

Watch this Scientific Journal Video about Depletion of Specific Cell Populations by Complement Depletion at JoVE.com

Depletion of Specific Cell Populations by Complement Depletion

効果的に浄化がしばしば必要とされる免疫細胞集団の機能を研究する。補体の枯渇は、高純度と免疫細胞集団を単離するための高速かつ安価な手法です。

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 ...

depletion-of-specific-cell-populations-by-complement-depletion

Research

JoVE Journal

Immunology and Infection

22.0K ビュー.  Blood Research Institute. 効果的に浄化がしばしば必要とされる免疫細胞集団の機能を研究する。補体の枯渇は、高純度と免疫細胞集団を単離するための高速かつ安価な手法です。

ビデオ: Depletion of Specific Cell Populations by Complement Depletion

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

免疫・感染学

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

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

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 ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

ウイルスの変異頻度のRNAウイルスと特性の忠実性の変異の分離

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

マウスのマクロファージの枯渇と再構成

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

デュアルカラータイム·ゲーティッド誘導放出枯渇（STED）ナノスによる免疫シナプスの可視化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

効率的なためキラー人工抗原提示細胞（KaAPC）<em&gt;体外</em&gt;ヒト抗原特異的T細胞の枯渇

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

マクロファージコレステロール枯渇との食作用への影響<em&gt;クリプトコックス·ネオフォルマンス</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

生成<em&gt;デ・ノボ</em&gt;センHemichainのレトロウイルス形質導入によって抗原特異的ヒトT細胞受容体

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

アクチンおよび誘導放出の枯渇 (STED) 顕微鏡を用いた B 細胞免疫シナプスにおける微小管細胞骨格の可視化

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...