Name: neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents
Uploaded: 2016-03-25T15:00:00
Description: Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

初代白血病細胞1.好中球の単離と共培養注：手順は、すべての患者がインフォームドコンセントに署名リヨン病院倫理委員会の承認の下で行いました。 <ol><li> 初代白血病細胞と好中球の単離 <ol><li>慢性リンパ性白血病（CLL）と診断された患者からEDTA（血液1ミリリットル当たり1.8ミリグラムのEDTA）に末梢血のチューブを収集します。 </li><li>滅菌50mlチューブ...

<ol>
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また、分離回収され、密度勾配遠心分離法を用いて高純度のヒト血液から好中球の単離のために、同じ手順単核細胞内で、ここに有効な、簡単、迅速かつ安価なプロトコルを記載しています。単離された細胞集団は、≥90％純粋です。 いくつかの方法がヒトの血液から好中球の単離のために用意されています。これらは、不連続勾配11,12を使用して、または好中?...

自然免疫細胞は、腫瘍微小環境内の細胞の本質的な割合を構成し、患者や癌1の動物モデルにおいて腫瘍の悪性度と関連しています。最近では、より広く、慢性免疫応答は、化学療法2の腫瘍の進行、転移および抵抗の促進に重要な役割を果たしていることは理解となっています。マクロファージは、直接化学療法3,4に対する腫瘍細胞の応答を調節することが示されてきた重要な自然免疫細胞です。しかしながら、抗癌治療に対する腫瘍応答の調節に好中球、先天性免疫系において重要なプレーヤーの役割は、不明です。これらのプロトコルの目的は、CLL患者の血液サンプルからの好中球を分離し、抗リンパ腫剤に対するリンパ腫細胞の感受性の調節におけるそれらの役割を研究するために、顆粒球経路に沿ったHL60細胞を区別するために、高速かつ信頼できる方法を使用することです。 6の侵入に対する防御の第一線としての血液5と行為における自然免疫系の最も豊富な細胞成分です。好中球は、いくつかの病的状態7に可変エフェクター機能に加えて、効果的な自然免疫応答を上昇中で重要な役割を持っています。したがって、このような密度勾配分離法などの他の血液細胞から好中球を単離するための迅速かつ信頼できる方法は、in vitro試験のために必要とされます。好中球の単離のために、このメソッドを使用すると、インビボおよびex vivo での好中球媒介性の免疫機能に関するさらなる研究を促進します。 好中球の純粋な集団を得る能力は、免疫学的疾患8を有する患者の調査のための重要な第一歩です。密度勾配分離法は、細胞の高い収率が得られる理想的な技術です。メソジスト教徒dは休憩なしで35分間300gで遠心分離した希釈したヒト血液を含む管の底に密度勾配溶液を添加することを含みます。単核細胞のリングは、インターフェイスに表示され、好中球は、前者の下に常駐します。この方法は、はるかに9高価である好中球単離キットなど、他の利用可能な方法に対して大きな利点を持っています。また、ヒト好中球に特異的な表面マーカーに対する抗体を用いて市販のキットによって、ヒト血液から好中球を単離し、細胞の活性化または分化のリスクを高めます。密度勾配分離法は、短時間での好中球の単離を可能にします。同じステップ内では、単核細胞はまた、分離回収されています。これは、純粋な細胞の高い収率を機能的完全性を実現するために取得された基本的な技術です。 腫瘍microenvを模倣するために、ironment、3D実験を行いました。 in vitroでの好中球の半減期が短い考えると、新鮮なヒト好中球と3D実験は決定的なものではありません。このため、前骨髄球（HL60）細胞を、分化誘導剤、ジメチルスルホキシド（DMSO）及びレチノイン酸（RA）を使用して好中球様細胞に分化するように誘導されます。分化したHL60細胞（HL60 デフ）を使用すると、異なるドナーからの単離に起因する好中球の異なる応答を持つ防ぐことができます。 試験管 3D で培養モデルは、in vitroでの2Dモデル間およびin vivoモデル中間段階を表します。 2D培養では、細胞は、この合成表面に変性された堆積タンパク質への不自然な細胞の添付ファイルを形成するプラスチック表面に広がります。逆に、細胞を、それらが合成細胞外マトリックスからの3D培養フォーム天然細胞間添付の細胞は、それらが結合している天然物質です。この理由のために、特に癌細胞および他の細胞型との間の三次元共培養モデルは、腫瘍増殖、血管形成、および転移への寄与を示すために非常に有用でした。その結果、三次元培養は、細胞培養は、 インビボ 10 に存在する生理学的条件を模倣します。

<table border="0" cellpadding="0" cellspacing="0" width="1161">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">RPMI 1640&#160;</td>
			<td style="width:157px;">Gibco Invitrogen</td>
			<td style="width:156px;">21875-034</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fetal bovine serum (FBS)</td>
			<td>Gibco Invitrogen</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Phosphate-buffered saline (PBS contains calcium and magnesium)</td>
			<td>Gibco Invitrogen</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td>
			<td>Life technologies</td>
			<td style="width:156px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin streptomycin (Pen Strep)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruton&#39;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&#160;</td>
			<td style="width:157px;">CliniSciences</td>
			<td style="width:156px;">A3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine&#160;</td>
			<td style="width:157px;">EG labo</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">BD Matrigel basement membrane matrix&#160;</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Put at 4 oC overnight before the day of the experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Red cell lysis buffer&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:156px;">555899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll (Pancoll)</td>
			<td>PAN Biotech</td>
			<td style="width:156px;">P04-60500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Retinoic acid</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">R2625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine serum albumin (BSA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td>A7906</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:156px;">E5134</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium chloride (NaCl)</td>
			<td>Euromedex</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Annexing V-FLOUS staining kit&#160;</td>
			<td style="width:157px;">Roche</td>
			<td>11&#160;988&#160;549&#160;001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kit RAL 555 Modified Giemsa staining kit</td>
			<td style="width:157px;">Cosmos Biomedical</td>
			<td style="width:156px;">CB361550-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometry</td>
			<td>BD Biosciences</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cytocentrifuge</td>
			<td style="width:157px;">Thermo Scientific</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMR-XA microscope</td>
			<td style="width:157px;">Leica Microsystems</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cellometer Auto T4 Cell Viability Counter</td>
			<td style="width:157px;">Nexcelom&#160; Bioscience</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

白いリングは、単核細胞を表し、血小板及び血漿：ここに記載の密度勾配分離法は、CLL患者の血液から単離した一次白血病細胞と非刺激好中球は、 図1Aは、上から下に（密度勾配遠心分離後に得られた異なる血液層を表し、提供します密度勾配溶液、顆粒球および赤血球）。 図1B及び1Cは、それぞれ好中球（マルチローブ核細胞）と単核細胞?...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Research

JoVE Journal

Immunology and Infection

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

ダブル勾配遠心、テフロンコーティングされた細胞培養バッグ中のマクロファージへの分化によるヒト単球の単離

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

細菌Pyomelanin産生を阻害し、酸化ストレスに対する感受性の増加に対応を決定するための方法

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

治療抗体間の標的結合およびCDC誘導を比較するためのインビトロ法：Biosimilarity Analysisにおける応用

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

アスペルギルス症では実験的肺真菌負担を決定する組織の定量化

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

ワクチン接種後のモルモット モデル メジャー T 細胞反応に最適化されたインターフェロン-γ しなさいアッセイ

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

多重蛍光免疫組織化学染色、イメージング、およびリンパ腫の組織学的試料中の分析

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...