この記事で報告された作品は、国立保健オーストラリアプログラムグラント（CRP）とNHMRCプロジェクト無償（CRPとBJCQ）の医学研究評議会（NHMRC）によってサポートされていました。またBJCQはNHMRCピータードハティー博士研究員です。

1）試薬のセットアップ 1.1）CFSEストック溶液 CFSE色素は、通常、25mgのバイアルに、カルボキシフルオレセインジアセテートスクシンイミジルエステル（CFDA、SE）（MW 557.47グラム/モル）として購入されています。 （数ヶ月まで-20℃で50〜100μL分注しのような店）の5mMの最終的なストック溶液のために8.96 mLのDMSOに25 mgを溶解する。それはそのような露出が貯蔵中に回避することが重要であるので、CFDA、SEは、水溶液と反応する。 1.2）リンパ球脾臓とやマウスからのリンパ節からリンパ球を分離し、0.5〜× 10 6の細胞濃度を5％熱不活化（HI）ウシ胎児血清（FCS）、を含む培地を1mL（例：RPMI）に再懸濁する- 10 × 10 7 / mLの。 2）CFSEラベリング 2.1）ルーチン方法： <ol class="lalpha"><li>徹底的に新鮮な（非接流体）を10mLのコニカルチューブの底に慎重に培地と場所の1mLの体積で細胞を懸濁します。 </li><li> （非接液チューブを使って移動し、途中でCFDA、SEのソリューションとの混合から1 mLの細胞懸濁液を防止する）水平にチューブを置きます。 </li><li>慎重に細胞溶液との接触をしない保証するチューブの上部にあるプラスチック製の非接液部分にPBS 110μLを加える。 </li><li> 110μLのPBSでCFDA、SEの5mM株式の1.1μLを再懸濁します。 </li><li>すぐにチューブにキャップをし、ソリューションの混合素早く均一に得るために井戸を反転し、渦。 CFSE色素は、5μMの最終濃度にあることに注意してください、しかし、最適な濃度は、調製した各バッチに対して決定する必要がある場合があります、そして、それが効果的であることを保証するために6ヶ月毎にチェック。 </li><li>室温で5分間細胞をインキュベート。 CFSEにCFDA、SEの変換（説明を参照）により色素は蛍光灯や過度の光に曝露することによって漂白のため、発生しやすくなることに注意してください。従って、それはアルミホイルでそれを覆うことにより、例えば、この点からの光からチューブを保護することをお勧めします。 </li><li> 20 ° Cと廃棄上清で5分間、300 × gで遠心分離により沈降、20℃PBS 5％HI FCSを含む10倍量に希釈して細胞を洗浄。さらに2回洗浄繰り返します。 </li></ol> 2.2）にも高い細胞数（10の適切な代替方法、 - 30 × 10 7）： <ol class="lalpha"><li>得るために井戸を1mlのPBSに5 mMストックの2μLを添加すると迅速に徹底的に再懸濁した細胞を1 mLにこれを追加することで2 ×（10μM）CFDA、SEのソリューションを準備し、その後すぐにチューブをキャップと反転し、渦素早く均一混合。 </li><li>室温で5分間細胞をインキュベートし、手順2.1（f）および2.1（g）のように洗浄。 </li></ol> 2.3）次に、細胞を細胞分裂の誘導のための関心のプロトコルに適用することはできません。標識された細胞は、in vitroおよび in vivoアッセイの両方で使用できます。 2.4）増殖アッセイの完了時に、細胞を採取していると（代表的な例については下記参照）フローサイトメトリーで分析した。 3）代表者の結果 CFSEの希釈によるリンパ球増殖を評価するために、それは分割されていない細胞（すなわち、最大蛍光）と非標識細胞（すなわち、最小の蛍光）の蛍光を知っておくと便利です。したがって、実験的なデザインが考慮に入れ、これらのコントロールを取る必要があります。下記のin vitroおよびフローサイトメトリーによってCD8 + T細胞の分裂を測定するためにCFSEを用いたin vivo試験の例である。 in vitro刺激 3.1） 図1は、のCFSEのプロファイルを示してCFSE標識オボアルブミン（OVA）特異的T細胞受容体トランスジェニックOVAの様々な量でパルスした樹状細胞と3日間の培養後のCD8 + OT - I T細胞。 CD8 + OT - Iマウスの脾臓とリンパ節から精製したT細胞が3.3 × 10 4 DCと共に培養CFSEと1 × 10 5細胞を標識した。 DCはここで、C 37℃で1時間OVAでパルスし、二回文化への事前の洗浄。 3日後、収穫し、標識抗CD8 - APC抗体とヘキスト33258とし、フローサイトメトリー（50,000件のイベントが収集された）によって分析した細胞。ライブは（ヘキスト33258 - ）CD8 +細胞が示されています。非標識細胞は自家蛍光細胞と検出可能な細胞分裂の限界のを示しながら、コントロールとして、単独で培養したCD8 + T細胞は、非分裂細胞のCFSEの強度を示しています。 4 CFSEピークは細胞が3分割まで受けていることを示し、この例ではパネルのそれぞれで見ることができることに注意してください。 in vivoで刺激3.2） 図2は、 生体内での 3日後にCFSEで標識したOVA特異的T細胞受容体トランスジェニックCD8 + OT - I T細胞のCFSEのプロファイルを示しています私■20μgのOVAの存在。 CD8 + CD45.1ジェニックOT - Iマウスの脾臓およびリンパ節から精製したT細胞をCFSEと5 × 10 6個の細胞は、C56BL/6Jの外側尾静脈（CD45.2 +）マウスに静脈注射で標識された。 2時間マウスの後にOVAを20μgと静脈注射した。 3日後、マウスから脾臓を抗B220 - PerCP、抗CD8 - APC - Cy7と抗CD​​45.1 - PE - Cy7抗体とヘキスト33258で標識された、単一の細胞懸濁液に加えた、収集して分析したフローサイトメトリーによる（1,000,000イベントが収集された）。ライブ（ヘキスト33258 - ）B220 -細胞は、右側のパネルに示すように左のパネルと+ CD45.1 +細胞をCD8ゲートに示されています。コントロールとして、無刺激のCD8 + T細胞をCFSE標識、非分裂細胞のCFSEの強度を示し、非標識細胞間、細胞および検出可能な細胞分裂の限界の自家蛍光を示しています。 CD45アロタイプ差を利用し、転送CD8 +彼らは合計CD8 + T細胞（左パネル）のマイナーなサブセットであるため、ホストからのT細胞を、解決に不可欠であることに注意してください。ほとんどの細胞は細胞が6部門にまで受けていることを示し、この例（右パネル）の7 CFSEピーク内に収まっていることに注意してください。 <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="図1" /> 図1。 CFSEで標識した卵白アルブミン（OVA）特異的T細胞受容体トランスジェニックCD8 OVAの異なる濃度でパルスしたDCへの試験管内で反応する+ OT - I T細胞、3日間培養した後であること示されているデータが。注意CD8の非分割人口+ T の能力抗原（ライトグレーのヒストグラム）と非標識の人口（濃い灰色のヒストグラム）の自家蛍光の有無で細胞。図は、CD8 +より高い抗原濃度で割るより多くのT細胞とCFSE希釈ピークに基づいて1〜3回に分けているT細胞を、示しています。 <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="図2" /> 図2。の能力をCFSEで標識した卵白アルブミン（OVA）特異的養子OVAに応答するためにC57BL / 6マウスに転送T細胞受容体トランスジェニックCD8 + OT - I T細胞、、養子移入後3日以内に示したデータ左パネル：。CD8 +、CD45。レシピエントマウスに1 + OT - 1 T細胞。右のパネル：CFSE細胞増殖プロファイル。 CD8の非分割人口の点に注意してください+レシピエントマウスのT細胞が抗原（ライトグレーのヒストグラム）と非標識リンパ球（濃い灰色のヒストグラム）の自家蛍光を受信して​​いません。図は、CFSE希釈ピークに基づいて1から6回を分けているCD8 + T細胞を示す。 <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="図3" /> 図3。カルボキシフルオレセインジアセテートスクシンイミジルエステル（CFDA、SE）を持つ細胞のラベリング中に発生する様々な分子事象の表現。プロセスの詳細については、ディスカッションのテキストを参照してください。注：一般的な頭字語&quot;CFSE&quot;ではなく、CFDA、SEは、一般の標識試薬とラベリング手順を記述するために使用されます。教区4に基づく図。

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

利害の衝突は宣言されません。

CFSEの仕​​組みと、なぜ急速な標識方法に感謝するために、増殖の解析での使用に必要な、それはCFSE細胞標識の分子基盤を理解しておくと便利です。これは二つのフェーズ、1）セルのエントリ、2）タンパク質の標識（図3）に分けることができます。 1）セルのエントリ：セルへの染料のエントリーは、染料のジアセチル形、カルボキシフルオレセインジアセテートスクシンイミジルエステル（CFDA、SE）によって媒介される。酢酸塩は、原形質膜を介してその迅速なフラックスを可能に色素性の高い膜透過性を作る。細胞内に存在するエステラーゼ、、それによって切断するCFDA、SEから酢酸とはるかに少ない膜透過性である染料のCFSEの形態を生じさせるには、このように細胞内の色素を集中。 2）タンパク質標識：CFDA、SEとCFSEの両方がアミノ反応性スクシンイミジル側鎖を持っているが、唯一のCFSEの蛍光灯です。 CFSEの高い細胞内濃度は、タンパク質を迅速かつ高いレベルの細胞内ラベリングを容易にします。細胞のCFSE標識は、代表的な結果（図1および2）に示すようにいくつかの部門を受けた細胞を解決するために重要である細胞の均一に標識された人口を、得るために迅速に実行する必要があります。

<!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>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminium foil</td>
<td>&nbsp;</td>
<td>Capral Aluminium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortex</td>
<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf (5810 R)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>LSR-II (BD Biosciences)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

カルボキシフルオレセインスクシンイミジルエステル（CFSE）はリンパ球分裂1-3を監視するために効果的で人気のある手段です。 CFSEは共有結合蛍光色素、カルボキシで長寿命の細胞内分子にラベルを付けます。このように、CFSE標識細胞が分裂するときに、その子孫は、カルボキシタグ付き分子の数の半分とを授けられており、したがって、各細胞分裂は、フローサイトメトリーを介して細胞の蛍光に対応する減少を測定することにより評価することができます。その低い細胞毒性と相まって非常に低分散の高い蛍光強度を持つリンパ球集団を、ラベルを付けるCFSEの容量は、その細胞分裂を測定するために理想的な色素こと。それは、フルオレセイン系染料なのでそれはまた、マルチカラーフローサイトメトリーにそれが適用されること、他の蛍光色素の広い範囲と互換性があります。この記事では、通常8細胞分裂にまで監視の目的のためにマウスのリンパ球を標識するために使用される手続きを説明します。これらの標識された細胞は、in vitroおよびin vivo試験の両方を使用することができます。

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSEは共有結合蛍光色素、カルボキシで長寿命の細胞内分子にラベルを付けます。このように、CFSE標識細胞が分裂するときに、その子孫は、それによって細胞分裂を評価するために使用できる蛍光の半分の量を、持っている。この資料では、通常CFSEでマウスのリンパ球を標識するために使用される手続きを説明します。

リンパ球増殖を監視するためのカルボキシフルオレセインジアセテートスクシンイミジルエステル（CFSE）の使用

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

the-use-carboxyfluorescein-diacetate-succinimidyl-ester-cfse-to

Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.3K ビュー.  John Curtin School of Medical Research, Australian National University. CFSEは共有結合蛍光色素、カルボキシで長寿命の細胞内分子にラベルを付けます。このように、CFSE標識細胞が分裂するときに、その子孫は、それによって細胞分裂を評価するために使用できる蛍光の半分の量を、持っている。この資料では、通常CFSEでマウスのリンパ球を標識するために使用される手続きを説明します。

ビデオ: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

運動誘発性リンパ球のアポトーシスの決定のための指先穿刺の採血方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

免疫・感染学

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS媒介細菌競争を監視するには、Visualアッセイ

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

活用<em生体内で&gt;</em新生児の実験マウスサイトメガロウイルス感染の進行を監視する&gt;イメージング

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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 ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Tリンパ球接着分子の相互作用の研究のための接着性の下でせん断応力の測定

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

の検出<em&gt;トリパノソーマ</em磁気活性化細胞選別し、フローサイトメトリーによって&gt;バリアント表面糖タンパク質の切り替え

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...