This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

ヒト血液サンプルを用いた研究がコンコルディア大学倫理審査委員会、証明書番号10000618.によってコンコーディア大学倫理審査委員会、証明書番号2010BERGによって承認されたマウス筋肉の作業を承認されました。 全血から1 PBMC単離注：無菌技術と製造業者滅菌装置を使用してバイオセーフティキャビネットには、この手順を行ってください。 <ol><li>慎重に50ミリリットルの滅菌コニカルチューブにヘパリン化（抗凝固剤）採血管からの全血を10〜15ミリリットル注ぐ。マイナー血液の流出の場合は、クリーニングティッシュを使用してのddH 2 O、70％EtOHで拭いてください。 </li><li>リン酸緩衝生理食塩水（PBS 1X）pHが7.4と1：1の比率にコニカルチューブ内の血液を希釈します。最大総容量は30ミリリットルを超えないようにしてください。 </li><li>気泡を避け血清学的ピペットガンを使用して穏やかに混合。 注：血aを避けるためにチューブを反転させて混合しないでください遠心分離中キャップとその後の浸透でccumulation。 </li><li>新しい50ミリリットル容量のコニカルチューブに、（ 材料および機器の表を参照）フィコール-パックの13ミ ​​リリットルを追加します。ラックにチューブを直立してください。 </li><li>トランスファーピペットを用いて、希釈した血液を取る、上部付近のチューブの内壁に転送ピペットチップをタッチします。ゆっくりと安定した圧力で、スクロースの層の上に血液層を形成する管の内壁に沿って血液を移す。すべての血液が転送されるまで、この手順を数回行います。 </li><li> 5に培地加速度を設定したスイングローターで700×gで30分間室温でチューブをしっかりと遠心チューブにキャップをし、ZEROに設定された減速。 </li><li>ゆっくりとチューブを取り出し、層を乱すことなく、バイオセーフティキャビネットにそれを取り戻す。 </li><li>慎重にPBS /プラズマAの間に置かれたPBMCが配置されているバフィーコート（薄曇った白層）を、収集、ndはフィコール層をトランスファーピペットを使用して。スクロース層を収集回避し、赤血球細胞層を乱さない。 </li><li>新しい50ミリリットルの滅菌コニカルチューブに軟膜を転送します。これは、すべてのPBMCを収集するステップ1.8を繰り返し数回を取ることができる。 </li><li> PBMCとのチューブにpH7.4のPBSを追加し、45ミリリットルのマークに埋める。徹底的にチューブを振る。ボルテックスしないでください。 </li><li> 1を洗う：9に設定された最大加速と減速の両方で480×gで15分間遠心操作は、円錐管の底にPBMCのペレットの形成を観察します。 </li><li>漂白剤の10ミリリットルプラスチックビーカーにPBS（上清）を捨てる。 </li><li>優しく凹凸面（ すなわち、空のラック）に対して「ラッキング」によって細胞ペレットを緩めます。ボルテックスしないでください。 </li><li>チューブに新鮮なPBS pHが7.4の25ミリリットルを追加します。複数の管が処理されている場合は、このステップで一緒にペレットをプールする。 </li><li>ウォッシュ2：遠心分離機480のXGのWiで12分間チューブ9に設定された最大加速と減速の両方目。 </li><li>漂白剤のスプラッシュプラスチックビーカーにPBS（上清）を捨てる。 </li><li>凹凸面にゆっくりと「ラック」（ すなわち、空のラック）。ボルテックスしないでください。 </li><li>チューブに新しいPBSの25ミリリットルを追加します。 <ol><li> 50μlの細胞を取り出し、生存能力カウント用のマイクロ遠心チューブに移す。 </li><li>穏やかに混合するには、上下パンブルー（生存能力染色）とピペットの等しい量（50μl）を追加します。 </li></ol></li><li> 10μLを取り出し、1ml当たり細胞の生存率をチェックするために、血球計数器に転送します。細胞/ mlの数を記録します。 </li><li>細胞の所望の量を取り出し、9に設定された最大加速と減速の両方で480×gで12分間遠心操作を行なう。 </li><li>漂白剤を入れたビーカーにPBS（上清）を捨てる。 </li><li>凹凸面にゆっくりと「ラック」（ すなわち、空のラック）。 8を追加チューブにPBSを0μlの。 </li></ol> 2. PBMCスライドを作る<ol><li>顕微鏡スライド上に細胞を80μlのを置きます。別のスライドの助けを借りて、ドロップを塗抹またはスライドの両端に40μlの各2滴を配置。 </li><li>乾燥するために生物学的安全キャビネット内でスライドしたままにします。つや消し側の鉛筆でスライドにラベルを付けます。 </li></ol> 3.スライド上のサンプルの修正<ol><li> 4.5ミリリットルの99％エタノール、37％ホルムアルデヒドの0.5ミリリットルを混合して固定液を準備します。 </li><li>スライドを乾燥した後、新たに作製した固定液2 mlを取り出し、スライドの表面全体を覆​​うようにスライドの上に注ぐ。 </li><li> 1分間のスライドで解決策をしておきます。水道水で1分間のスライドをすすぎ、空気乾燥に任せる。 </li></ol> 4.ネガティブコントロールのためのアミラーゼのソリューションを作る<ol><li>アミラーゼ粉末を0.25gを取り、ジ50mlに溶解静め水。清潔な100mlビーカー内の溶液を注ぐ。 </li><li>スライドの半分は治療を受け、残りの半分は、未処理のままにした後、室温（ 図1C）で15分間インキュベートするようにビーカー中でスライドを浸す。 </li><li>アミラーゼ治療を受けているスライドのどちら側にメモしておいてください。治療群と対照との間の境界を示すスライドの背面に線を引きます。 </li><li>アミラーゼ溶液を除去し、空気乾燥したスライドを残してのddH 2 Oでスライドを洗浄します。 </li></ol> 5.過ヨウ素酸シッフ（PAS）染色および撮影を行う NOTE：PAS試薬は吸入により毒性があり、腐食性であるため、ステップは、化学ドラフト内で行われる必要があり、廃棄物が適切に機関のガイドラインに従って廃棄しなければならない。 <ol><li>平らな面にスライドを置き、試料上で定期的な酸溶液の1.50〜2.00ミリリットルを注ぐ。 Incuba室温で5分間テ。 </li><li>蒸留水のいくつかの料金のスライドを洗浄します。 </li><li>スライドにシッフ試薬の1.50〜2.00ミリリットルを注ぎ、15分間室温で静置する。 </li><li> 5分間蒸留水でスライドを洗浄し、空気乾燥に任せる。 </li><li>スライド上の10μlの取り付けメディアを適用し、二つの小さなカバーガラスでカバーし、または50μlのを適用し、一つの大きなカバーガラスを使用しています。 </li><li>一晩乾燥させて、カバースリップの端に明確なマニキュアを適用します。 </li></ol> 6. 100X対物レンズを用いて双眼光学顕微鏡で画像を得る

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The authors have nothing to disclose.

このビデオ記事の重要なステップは、細胞の洗浄およびアミラーゼ治療中であった。スライドを洗浄しながら、重要なステップは、プラスチック製のスクイーズ洗浄瓶を使用して、水が軽くスライド上のサンプルを介して実行させると、サンプル上に直接狙っていなかった。少しでも直接水圧は、細胞がスライドをオフに来てしまいます。もう一つの重要なステップは、±アミラーゼ条件に?...

過ヨウ素酸シッフ（PAS）染色は広く、筋肉の研究および診断に使用されている免疫組織化学的手法である。また、血液サンプルの診断ツールとして利用されている。技術は無色シッフ試薬、それにより深いマゼンタ生成物を生成物と反応アルデヒド基を生成する多糖類内のユニットを酸化したサンプルに過ヨウ素酸ナトリウム溶液を適用することによって機能する。この手順のステップは、図1に示されている。汚れが多糖部分を有するグリコーゲン、糖タンパク質、糖脂質、ムチン、または他の分子を含む多糖類マゼンタで何をオン。 PAS染色は、多くの場合、筋線維におけるグリコーゲンレベルを測定するために使用される。筋肉組織切片彼らはしっかりとスライドに付着し、複数の洗浄と染色工程に耐えるような技術に最適です。グリコーゲンは、高い需要があり、タイプII筋線維、速筋の中で最も存在する最大パフォーマンス1,2のためのグリコーゲンを必要とする迅速なATP産生のために。グリコーゲンは、グリコーゲンホスホリラーゼ酵素の作用を介して遊離グルコースに分解することができるグルコースの分岐ポリマーである。栄養不足や高エネルギー需要の時代にしながら、休息と栄養自給の時代には、グリコーゲンは、グリコーゲン合成のプロセスを経て補充される。グリコーゲンは、グリコーゲン分解によるグルコースに分解される。血液サンプル上で、早ければ1950年の臨床医科学者が調査してきたように、PAS染色からは、様々な疾患3-7のグリコーゲン含有量を分析する。例えば、ポンペ病、真正グリコーゲン貯蔵において、疾患白血球は健常対照8と大きく異なるグリコーゲンを大量に蓄積する。 このビデオ資料は、末梢血単核細胞（PBMC）健常なヒト被験者の静脈血からのサンプル上で使用するためのPAS染色の適合したバージョンを示す。 PBMCsは主にTリンパ球のリンパ球およびBリンパ球のファミリー、ならびにナチュラルキラー細胞および単球のような他の免疫細胞を含む。最初の精製工程は、赤血球、好中球、および他の顆粒球を除去する。この技術は、全血塗抹標本を使用する場合と比較して、PAS陽性細胞のより堅牢な列挙を可能にするリンパ球の濃縮された割合のデータを提供する。 <img alt="図1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> 図1：PBMC上のPAS染色の段階の方法論によって、工程（A）まず、PBMCの単離はFicoll勾配を介して達成されるが、左側のパネルには、遠心分離前の準備を示し、右側のパネルには、遠心分離後にそれを示してPBMCを含む軟膜チューブの中心に観察される。（B）単離されたPBMCは、ホルマリン、エタノール固定剤ソリューを用いてスライド上に固定されているる。スライドを穏やかにプラスチック洗浄瓶からの蒸留水で洗浄する。（C）スライドは、次に、グリコーゲンを溶解するアミラーゼ溶液で満たされた100mlビーカー途中に配置される。スライドを穏やかにリンスした。（D）スライドは糖の酸化が起こる周期的な酸溶液で処理される。スライドを軽くすすぐ。これは、過剰の過ヨウ素酸を除去し、酸化工程を停止します。（E）シッフ試薬をスライドに添加される場合には、酸化工程中に作成されたアルデヒドと反応する。この無色の試薬は、その後、深い赤マゼンタ製品になります。スライドを静かに過剰シッフ試薬を除去するために洗浄する。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

試薬及び基本技術を検証するために、PAS染色をマウスヒラメ筋切片に、製造業者の指示に従って行った。染色は犠牲と同じ日に行われた染色の最後のステップで、切片をキシレン中で固定した。ヒラメ筋は9〜35％のグリコーゲン陽性細胞を含むことが知られている。染色された筋細胞は、2つのセル内の異なるPAS陽性と特長点状顆粒、および細胞膜（ 図2A）の境界を定め?...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

Research

JoVE Journal

Immunology and Infection

21.0K ビュー.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

ビデオ: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

共刺激シグナル遮断によるアロ抗原刺激によるヒト末梢血単核球のアロ抗原特異的アネルギーの誘導

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

免疫・感染学

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  &amp;#947;&amp;#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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用してリンパ球血管外漏出の分析<em&gt;インビトロ</em&gt;ヒト血液脳関門のモデル

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

感染性単核球症の小児からの末梢血サンプル中の免疫細胞亜集団の分離

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

マイクロバブルを用いたヒト末梢血単核細胞サンプルからの浮遊選鉱ベースのT細胞の単離、活性化、および増殖(英語)

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

ミトコンドリア生体エネルギー分析のためのPBMCコレクションと凍結保存

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

著者スポットライト:凍結保存後の末梢血単核細胞における生体エネルギーパラメータの保存 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

ヒト末梢血単核細胞の凍結保存と生体エネルギー評価

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...