Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

私たちは、この作業に参加する意欲のためにマイアミWIHSの研究参加者に感謝します。この原稿のデータは、マイアミの女性の省庁間HIV研究（WIHS）により回収しました。本書の内容はもっぱら著者の責任であり、国立衛生研究所（NIH）の公式見解を示すものではありません。この作品は、前進のための国立アレルギー感染症研究所と女性の省庁間のHIV感染の研究（WIHS）[助成金番号U01のAI103397]、アレルギーと国立感染症研究所、国立衛生研究所[助成金番号P30AI073961]、ナショナルセンターによってサポートされていましたトランスレーショナル科学とマイノリティの健康と健康格差に国立研究所、国立衛生研究所[助成金番号UL1TR000460と1KL2TR000461]、および国立小児保健発育研究所、国立衛生研究所[承認番号K23HD074489]。

研究活動は、マイアミの女性のHIV省庁間研究（WIHS）と承認が前に得られたエイズ研究のためのマイアミセンター（CFAR）.Institutional審査委員会（医学のマイアミミラー科大学）と共同で大学マイアミのHIV研究ユニットで開催されました募集及び任意の評価や研究関連手順へ。 1.子宮頸管ブラシサンプル採取注：参加者は、訓練を受けたMDや婦人科医と細胞採取用ブラシを挿入することにより、鏡検査下で実施した上皮内リンパ球の集合によって行わ膣の検査を受けました。 <ol><li> 参加者の特徴 <ol><li>性的に、アクティブ妊娠していないではなく、避妊薬上または子宮内のデバイスとの年齢、18〜45年を老化させる女性の参加者を募集。 </li><li>膣の検査の前に、参加者は緑で10 mLの採血を受けています末梢血単核細胞（PBMC）を単離するためのトップヘパリンバキュテナー。子宮頸管上皮内分析9の制御として機能します。 </li></ol></li><li> 子宮頸管サンプルの収集 <ol><li>無給油で膣内に適当なサイズの滅菌鏡を挿入します。粘液をきれいに検鏡し、滅菌ガーゼの挿入を容易にするために暖かい水を使用してください。鏡の位置は、OSや子宮膣部の完全な可視化を可能にします。 </li><li>手に最も近いだけ毛が表示されるまで、子宮頸管に子宮頸管ブラシを挿入します。 360°用のブラシを時計回りに回転させます。 IMDM培地5mlを含む15 mLのチューブにブラシを転送します。 </li><li>すぐに収集した後、氷上で細胞採取用ブラシを含むチューブを置きます。収集した上皮内細胞の高い生存率を得るためには、1時間以内に研究室およびプロセスへの細胞採取用ブラシを提供します。 </li></ol></li></ol><ol><li> 4短い（10秒）ストロークを適用頸管細胞採取用ブラシを含む渦管。 </li><li> 250×gで10分間、細胞採取用ブラシを含む遠心チューブ。 </li><li>慎重にチューブから細胞採取用ブラシを削除する（チューブの底にペレットを乱さないでください）、IMDM培地の1ミリリットルを追加し、チューブを氷上に置きます。 </li><li> 50mlのチューブの上に100ミリメートルのセルストレーナーに細胞採取用ブラシを置きます。 </li><li> 20ミリリットルのIMDMで細胞採取用ブラシを洗ってください。 </li><li> 250×gで10分間遠心分離管。 </li><li>上清を除去し、ペレット化した細胞を1mLのIMDMに再懸濁し、2.3に記載され、すでに再懸濁ペレットと組み合わせます。 </li><li>自動細胞計数器10を用いて細胞数をカウントしたり、トリパンブルー色素の10μLでの細胞懸濁液10μLを組み合わせることにより、血球計数器を用いて細胞数を計測します。優しく細胞と色素を混合するまでピペット上下10倍。負荷10μL2血球計数器のチャンバのいずれかの開口部に混合物の。 5グリッド領域内のすべてのセルを数えます。実行可能な非生存細胞の別々のカウントを保持。以下の式を用いて細胞生存率を決定する：％生存細胞=非生存細胞×100 /総細胞数の細胞。 以下の計算を使用して、1ml当たり、その後の細胞濃度（細胞の合計数）を決定します。 1ml当たりの全細胞は=全細胞カウントX（希釈係数/カウント正方形の数）は10,000細胞/ mlにxは。 </li><li> 10％DMSOおよび90％FBSを含む凍結培地を用いて、単離された子宮頸管細胞を凍結します。 </li></ol> 3.フローサイトメトリー<ol><li>再懸濁1％ウシ血清アルブミンを含有する100μLのFACS緩衝液中に1-3×10 5子宮頸管細胞（PBS </li><li> 1μL/ 10 6 表1に記載の濃度で表面マーカーのためのLIVE / DEAD修正可能イエロー死細胞染色キットおよび抗体を追加します。 </li><li>光から保護して4℃で30分間、細胞をインキュベートします。 </li><li> 350×gで5分間、1mLのFACS緩衝液細胞および遠心管を追加</li><li> 300μLの1％パラホルムアルデヒドで細胞ペレットを再懸濁します。 </li><li> FACS緩衝液100μLビーズの一滴、サンプル染色のために使用される抗体と同じ濃度を添加することにより、染色に使用されるビーズと抗体を使用して補正制御を準備します。 </li><li> 405 nmで、488 nmの、および635nmのレーザーを搭載したフローサイトメーター、上の染色されたビーズとサンプルを取得します。 </li></ol> 4.子宮頸管ガンマデルタT細胞の単離<ol><li> 磁気ビーズ分離 <ol><li>再懸濁材料および試薬の表で参照TCRガンマデルタマイクロビーズキットに含まれるバッファの40μLで、2.6で説明した子宮頸管細胞ペレット（≤107総細胞）を、。 </li><li>まず、ウィット：2段階の染色プロトコールのためのキットの指示に従ってください抗ハプテンマイクロビーズ-FITCを有する第二のH抗TCRガンマデルタハプテン - 抗体と、 </li><li> 4~8℃で15分間インキュベートした後、10分間、300×gで遠心分離緩衝液の1ミリリットルを加えることによって細胞を洗浄します。チューブを反転させて上清を除去し、一枚の紙の助けを借りて乾燥タップします。 </li><li>提供バッファーの500μLで細胞ペレットを再懸濁し、ベンダーのプロトコルに従って適切な磁気分離器に置くことで磁気カラムを準備します。 </li><li>カラムに細胞懸濁液を適用し、通過する非標識細胞を収集し、バッファの適切な量（500μL）でカラム3回洗います。列のリザーバが空になると、バッファたびに3回を追加することにより、洗浄工程を実行します。総流出物を収集します。これは非標識、負の細胞画分であります</li><li>列を削除するカラムに磁気分離器およびピペット1mlの緩衝液を形成し、直ちに磁気ラベルで端数を洗い流しますしっかりカラム付属のプランジャーを適用することによって細胞を編 注：磁気標識された抗ハプテンマイクロビーズを蛍光色素FITCとコンジュゲートされているため、単離されたガンマデルタT細胞は、すでに蛍光フローサイトメトリー分析のために染色されています。 </li></ol></li><li> 細胞選別 <ol><li>セクション3で説明したように細胞選別のために、生存能力のダイと抗体パネルと子宮頸管細胞を標識。 </li><li>ライブ、CD45 + CD3 +、ガンマデルタ陽性細胞上の電子ゲートを設定し、細胞選別を行います。 </li></ol></li></ol>

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著者らは、利害の衝突をもたらす可能性がありコマーシャルまたは他の関連を持っていない（ 例えば 、製薬株式所有権、コンサルティング、アドバイザリーボードのメンバーシップ、関連特許、または研究資金）。

女性の下部生殖管の粘膜の脆弱性の評価は、HIV取得送信のリスクに対処する臨床試験の必須成分です。典型的には、HIV感染にFGTの脆弱性が性器分泌物（サイトカイン、ケモカインおよび抗菌ペプチド）12、13中の可溶性免疫調節を測定することによって評価されます。しかし、これらのバイオマーカーは、膣の炎症しか示している生理学的および病?...

我々は、上皮内GD T細胞を評価するために、子宮頸管ブラシサンプルを使用する方法を開発しました。子宮頸管は円柱上皮の単層によって裏打ちされています。上皮内リンパ球は急速に病原性微生物7、8に遭遇すると、免疫応答を開始することができ、上皮層内に存在する現場のリンパ球を表します。子宮頸管上皮内コンパートメントの免疫学的事象を理解することは、HIVを含む感染を防止するための効果的な戦略の設計のために重要です。 私たちの方法は、さらに、HIV感染またはHIV感染のリスクがある女性では上皮内GD T細胞の役割を探求するために新鮮に採取したヒト子宮頸管のサンプルと同様に凍結した子宮頸管細胞に適用することができます。このプロトコルの主な目的は、子宮頸管上皮内コンパートメントにおける新規免疫学的事象を発見し、evaluatことですHIV感染女性における粘膜の脆弱性のマーカーとしての電子GD T細胞応答。 FGTの免疫周りの知識の大幅なギャップが部分的に成功した粘膜サンプルを収集し、処理の難しさに起因しています。また、複雑な粘膜免疫細胞の不均一性、および互いにそれらの相互関連性は、免疫系の状態と機能を反映する臨床的に関連する測定値を識別することに大きな課題です。我々はさらに、FRT免疫の基礎となるメカニズムを同定するために重要であり得る非常に多重化され、単一細胞技術を用いて分析することができる高度に精製された上皮内GD T細胞を得るための方法を開発しました。

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

女性の生殖器官（FRT）粘膜免疫系は防御の第一線として機能します。性器粘膜のより良い知識は、HIVを含む異なる病原体の病原性を理解するために不可欠です。ガンマデルタ（GD）T細胞が「非在来型」T細胞の原型であり、γおよびδ鎖からなるヘテロ二量体、T細胞受容体（TCR）の発現によって定義されるT細胞の比較的小さな部分集合を表します。これは、α-βのTCRによって定義される古典的なはるかによく知られているCD4 +ヘルパーT細胞およびCD8 +細胞傷害性T細胞から離れてそれらを設定します。 GD T細胞は、多くの場合、組織特異的局在化を示し、上皮に濃縮されます。 GD T細胞は、炎症、腫瘍監視、感染性疾患、および自己免疫における免疫応答を調整します。 ここでは、再現性のヒト子宮頸管上皮内GD Tリンパ球を分離し、分析するための方法を提示します。我々ヘクタール女性の省庁間のHIV感染に関する研究（WIHS）に参加した女性から使用子宮頸管細胞採取用ブラシサンプルVEの。膣粘膜の傷害が存在である条件中GD T細胞相互作用についての知識は、粘膜GD T細胞応答についての知識があり、膣microbicides.In追加の開発を含む、粘膜の脆弱性に対処している任意の臨床試験に適用することができます感染性疾患の治療におけるGD T細胞ベースの免疫療法の適用のための潜在。

最近、我々は子宮頸管上皮内GD T細胞を分析し、我々はHIVに感染した女性では子宮頸管ガンマデルタT細胞の喪失9、11について報告する最初のものでした。ここでは、隔離し、子宮頸管ガンマデルタT細胞のサブセットを分析するためのプロトコルについて説明します。 図1に示すように 、GD T細胞は、...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.7K ビュー.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ビデオ: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

免疫・感染学

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

人間頸管サンプルの採取、単離、およびフローサイトメトリー分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

医学

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

単離と<em&gt;エクスビボ</emVδ1の&gt;文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT細胞、胸腺外αβT細胞前駆

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

神経膠腫浸潤末梢血単核細胞の単離およびフローサイトメトリー分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

イメージングフローサイトメトリーによるヒト単球由来樹状細胞の特徴付け：二つの単球の分離プロトコルの比較

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

前処理腫瘍細胞の同種共培養環境におけるヒトT細胞活性の解析

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

がん研究

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

7 免疫細胞サブセット 2 螢光色素の差別フローサイトメトリー

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

フローサイトメトリーによる分析のための子宮内生リンパ球細胞の分離

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

CD4 の分離+ T 細胞および循環の解析 T 濾胞ヘルパー (cTfh) 末梢血を使用して 6 色フローサイトメトリーから細胞のサブセット

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...