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).

1.中性粒细胞分离共培养原发性白血病细胞注：过程是里昂医院伦理委员会与签署知情同意所有患者同意下进行的。 <ol><li> 原代白血病细胞和中性粒细胞的分离 <ol><li>从确诊为慢性淋巴细胞白血病（CLL）的患者收集EDTA（每毫升血液1.8毫克EDTA）外周血管。 </li><li>各15毫升的血液添加到无菌的50ml管中，并用15毫升的RPMI稀释（稀释1：1），然后小心地慢慢加入溶于15...

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这里所描述的，有效的，简单，快速和廉价的协议为嗜中性粒细胞的人血液中分离，使用密度梯度离心的方法的高纯度和相同的步骤的单核细胞内也分离和回收。所述分离的细胞群是≥90％的纯度。 有几种方法可用于从人血液嗜中性粒细胞的隔离。这些包括类似的方法使用连续梯度11,12，或使用嗜中性粒细胞分离试剂盒由阳性免疫磁性选择期间的中性粒细胞是免疫磁?...

先天免疫细胞构成的细胞的肿瘤微环境内的重要比例，并已与患者和癌症1动物模型中的肿瘤恶性相关。最近，已变得更加广泛地理解，慢性免疫应答中促进肿瘤进展，转移和耐化学疗法2发挥关键作用。巨噬细胞是已被证明直接调节对化疗3,4-肿瘤细胞应答重要的先天免疫细胞。然而，中性粒细胞，在先天免疫系统的主要参与者的作用，在调节肿瘤响应于抗癌治疗是不知道。这些协议的目的是使用快速和可靠的方法来分离来自CLL患者的血液样本的中性粒细胞和分化的HL60细胞沿粒细胞途径，以研究在调节淋巴瘤细胞抗淋巴瘤剂的敏感性的作用。 6在血液5，并作为防御的第一道防线是先天免疫系统的最丰富的细胞成分。嗜中性粒细胞有几种病理条件7上升除了可变效应子功能有效先天免疫反应中起重要作用。因此，以分离从其它血细胞，如密度梯度分离法中性粒细胞的快速和可靠的方法，需要在体外研究。使用这种方法的中性粒细胞隔离将有利于在体内和体外中性粒细胞介导的免疫功能进一步研究。 以获得嗜中性粒细胞的纯群体的能力对于患者的调查与免疫疾病8的重要的第一步。密度梯度分离方法是在其中获得的细胞高产率的理想的技术。该方法具ð涉及在含有稀释的人血，然后离心以300g为无休息35分钟试管底部加入密度梯度溶液中。的单核细胞的环出现在界面和嗜中性粒细胞驻留低于前者。这种方法具有相对于其他可用的方法显著优点，如嗜中性粒细胞分离试剂盒这是更昂贵的9。此外，通过使用针对特异于人嗜中性粒细胞的表面标志物的抗体的商品试剂盒中分离人血液嗜中性粒细胞，增强细胞活化或分化的风险。密度梯度分离方法允许嗜中性粒细胞的一个短的时间周期内的隔离。在同一步骤，单核细胞也分离和回收。它是在其中，以实现功能完整性得到纯的细胞的高收率的基本技术。 为了模仿肿瘤microenvironment，进行3D实验。定体外中性粒细胞的半衰期很短，用新鲜的人中性粒细胞的3D实验不是决定性的。出于这个原因，早幼粒细胞（HL60）细胞被诱导分化成使用分化诱导剂二甲基亚砜（DMSO）和视黄酸（RA）嗜中性粒细胞样细胞。用分化的HL60细胞（HL60 差异 ）将防止有中性粒细胞，由于来自不同供体隔离不同的反应。 体外三维培养模型代表在体外 2D模型之间和体内模型中的中间阶段。在二维培养，细胞扩散塑料表面上形成不自然的细胞附件被这个合成表面沉积变性的蛋白质。相反，在由于细胞和它们合成的细胞外基质的3D培养形式自然细胞 - 细胞附件细胞对它们所连接的天然材料。出于这个原因，3D共培养模型，尤其是癌症细胞和其他细胞类型之间，已经指示他们的肿瘤的生长，血管生成和转移的贡献非常有用的。其结果是，3D培养使细胞培养模仿存在于体内 10的生理条件。

<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.

这里所描述的密度梯度分离方法提供了从CLL患者的血液中分离的原代白血病细胞和未刺激的嗜中性粒细胞图1A表示密度梯度离心后获得的不同血液层（从上到下：血小板和血浆，白圈表示的单核细胞，密度梯度溶液，粒细胞和红细胞）。 图1B和1C分别示出嗜中性粒细胞（多叶形核细胞）和单核细胞之间的形态出现的差异。 <p class="jove_content" fo:kee...

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.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

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 ...

The overall goal of these procedures is to quickly isolate highly purified neutrophils from human blood samples by density gradient centrifugation to investigate their role in lymphoma cell sensitivity to cancer therapy using two D and three D cell culture models. These methods can help answer key questions about the interactions between the innate immune system and cancer such as what role does a neutrophil mediated tumor cell responses play in cancer therapy. The main advantage of density gradient separation is the ability to isolate pure immune cell populations in a short period of time without exposing the cells to function altering stimuli.To isolate the primary leukemic cells and neutrophils, begin by adding 15 milliliters of EDTA treated blood from chronic lymphocytic leukemia patient samples into individual, sterile 50 milliliter tubes. Dilute the blood with 15 milliliters of RPMI. Then carefully and slowly add 15 milliliters of room temperature density gradient solution under the blood, taking care not to mix the layers.After separating the cells by centrifugation, four distinct phases will be visible. The platelets and plasma at the top, a white ring of mononuclear cells, the density gradient solution, and the granulocytes and erythrocytes at the bottom. Using a plastic pasture pipette, first transfer the white ring of primary leukemic cells in a new 50 milliliter tube.Wash the cells with up to 50 milliliters of PBS with calcium and magnesium. And re-suspend the palate in five milliliters of fresh PBS. Then, add 45 more milliliters of PBS to the cells, and spin down the cells again.After the second wash, re-suspend the palate in complete RPMI medium. And count the number of viable primary leukemic cells. Next, remove the platelet plasma and density gradient layers, and add 25 milliliter of PBS and 25 milliliters of Dextrin in sodium chloride to the granulocyte erythrocyte phase.Mix the tubes ten times by inversion. Then, let the cells settle for 30 minutes at room temperature. At the end of the equilibration, transfer the upper red blood cell poor neutrophil layer into a sterile, 50 milliliter tube and spin down the cells.Resuspend the palate in five milliliters of PBS and lyse the red blood cells with 45 milliliters of red cell lysis buffer. After 15 minutes in the dark at room temperature, spin down the cells again and wash the palate in 50 milliliters of PBS. At the end of the wash, re-suspend the cells in the complete RPMI medium and count the number of viable neutrophils.Dilute both groups of cells to a 300, 000 cell per 50 microliters of RPMI concentration and divide the samples between the appropriate number of five milliliter fax tubes. After centrifugation, resuspend the pellets in 50 microliters of PBS supplemented with FBS. To determine the purity of the isolated cell subsets, label the aliquant of primary leukemic cells with anti humanCD19 antibody conjugated to APC and the aliquant of the purified neutrophils with a mixture of anti human antibodies as listed in the table for 30 minutes in the dark at four degrees Celsius.At the end of the incubation, wash the cells in 500 microliters of FBS supplemented PBS and resuspend the palates in 200 microliters of fresh FBS PBS. Then, analyze the purity of the cell populations by flow cytometry, taking care to color compensate the samples. To co-culture the primary leukemic cells with the autologist neutrophils, see 200, 000 cells per milliliter of leukemic cells alone or with autologist neutrophils in a 24 well plate in complete RPMI medium.Mix the cells carefully. Then treat the cells with Brutton's Tyrosine Kinase Inhibitor for 24 hours at 37 degrees Celsius and five percent CO2. The next day, collect an aliquant of the cells in a five milliliter plastic fax tube.Then, spin down the cells and wash the pellets in one milliliter of FBS PBS. After the second centrifugation, label the cells aninexine five and propidium iodide according to the manufacturer's instructions for ten minutes at room temperature in the dark. Then, analyze the neutrophils and primary leukemic cells by flow cytometry using the illustrated gating strategy.To set up a three D co-culture, add 50, 000 RL lymphoma b-cells alone or mix with HL60 differentiated cells in sterile 15 milliliter tubes. Spin down the samples and resuspend the pellets in 300 microliters of basement membrane matrix, using a one millimeter pipette tip with the opening cut to two to three millimeters. Next, incubate 300 microliters of the cells in each well of a 24 well plate for 30 minutes at 37 degrees Celsius and five percent CO2.At the end of the incubation, add one milliliter of complete RPMI medium to each well and return the threeD cultures to the incubator for another seven days with medium changes every two days. On day five, add ten nanomolar of incristine to the cultures. After one week of culture, aspirate the medium and wash each well two times with one milliliter of ice cold PBS.Then, add three milliliters of ice cold PBS with EDTA to each well and scrape the bottom of the wells with a 200 microliter pipette tip to detach the gels. Shake the plate gently on ice for 30 minutes. Then, transfer the cell suspensions to individual sterile 15 milliliter tubes for gentle shaking on ice for another 30 minutes.Confirm the appearance of a homogenous cell suspension. Then, spin down the cells followed by a PBS wash and resuspend the pellets in fresh PBS with FBS. Finally, label the cells with the appropriate antibodies and viability dyes as just demonstrated and analyze the RL and HL60 differentiated cells by flow cytometry using the illustrated gating strategy.Analyzing all the events of the isolated cell populations by flow cytometry demonstrates that this separation method results in a highly pure single peak CD19 expressing primary leukemic cell population. And a greater than 90 percent pure neutrophil population positive for the leukocyte common antigens CD45, and the CD15 and CD16 antigens expressed by mature neutrophils. Gaiting on the primary leukemic cells from neutrophil co cultures demonstrates that the neutrophils exert a protective effect on cell survival against the tyrosine inhibitor ibrutinib with a significantly higher number of viable primary leukemic cells compared to the culture of primary leukemic cells alone.Differentiated HL60 cells are more stable in three D cultures than neutrophils. After induction of their granulocytic development, these cells are smaller than undifferentiated HL60 cells and express higher levels of CD11B and CD38. Differentiated HL60 cells also exhibit multi lobed nuclei.These cells exhibit a protective effect on the RL cells with a significantly higher number of viable RL cells in three D co cultures after vincrostin treatment compared to vincrostin treated lymphoma cells cultured alone. Once mastered, the density gradient centrifugation method can be completed in 90 minutes. After their development, these techniques paved the way for researchers in the field of cancer biology to explore the impact of various white blood cells on tumor malignancy in preclinical models and patient samples.After watching this video, you should have a good understanding of how to quickly and reliably isolate mononuclear cells and the neutrophils, analyze the effect of neutrophils on mediating tumor cell responses to anti cancer agents, and set up a three D co culture.

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

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

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

科研

免疫与感染

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

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

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

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

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

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

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

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

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

体外方法比较标的结合和CDC诱导治疗性抗体之间：应用在Biosimilarity分析

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

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</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

实验性曲霉病肺真菌负担的组织学定量测定

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

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

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

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

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, ...