Name: isolation and characterization of neutrophils with anti-tumor properties
Uploaded: 2015-06-19T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Neutrophils with Anti-Tumor Properties at JoVE.com

ZG支持从以色列科学基金会（批准号:41/11）的I-CORE项目赠款，在Abisch-福仁基金会，Rosetrees信托，以色列癌症研究基金会（ICRF - 研究职业发展奖）和令人关注的基础。 ZGF支持从以色列癌症研究基金会（ICRF - 研究职业发展奖）赠款，以色列卫生部和以色列肺脏协会的首席科学家。

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The authors have nothing to disclose.

嗜中性粒细胞是最丰富的全白血细胞，并且是在感染和炎症的情况下，第一反应者。因此，它们是外部线索高度敏感，并且容易激活。此外，嗜中性粒细胞具有非常短的半衰期和快速周转。总之，这些特点提高一些困难与中性粒细胞，这种独特的实验策略是必需的工作。例如，有几个中性粒细胞纯化策略，每一个都有自己的优点和缺点。 在中性粒细胞工作的一个关键步骤是从全血中的净化。嗜中性粒细胞可使用任一密度梯度或基于抗体的策略（阳性或阴性选择）被有效纯化。我们选择的方法是使用密度梯度的，因为它会产生高数量的高度纯化的嗜中性粒细胞具有最小的非特异性活化。然而，正如我们展示在最 ​​近的研究中21，机智^ h肿瘤进展中性粒细胞聚集在高数量的单核低密度部分。在这些条件下使用密​​度梯度提供了一种高纯度的高密度中性白细胞部分，这并不表示整个循环嗜中性粒细胞的曲目，而且是严重污染的其他单核细胞（淋巴细胞和单核细胞）低密度中性白细胞部分。在这些情况下选择的方法是一种基于抗体的纯化，优选负选择。使用抗体的纯化的中性粒细胞产生高纯度的中性粒细胞和更好地代表了整个循环嗜中性粒细胞的剧目。然而，我们注意到，在较长的中性粒细胞一起孵育的抗体，非特异性激活的机会增加。因此，我们建议，对于最佳结果，基于抗体的嗜中性粒细胞的纯化应尽可能快地进行。基于抗体的嗜中性粒细胞的纯化也是当选择浦的方法rifying从组织或肿瘤中性粒细胞。 无论选择哪种纯化过程，纯度，活力和功能的完整性，必须严格评估。嗜中性粒细胞的纯度通过流式细胞术使用以嗜中性粒细胞表面标记物反应的抗体来确定。在小鼠，LY-6G是特异于嗜中性粒细胞，其特征在于细胞CD11b +莱曼6C 低莱曼6G + F4 / 80 -表型。人中性粒细胞不表达的标记类似于到Lym-6G，并且通常特征在于的CD11b，CD15，CD16，和CD66b的表达。由于嗜中性粒细胞具有Fc受体，这些需要免疫染色前被阻塞。嗜中性粒细胞，也可以从其他的白血细胞由具有更高的SSC区分。生存力应在纯化过程的结束来确定（台盼蓝，协议2.1），并应大于98％一致更大。功能完整应得到普里确定fication从天真的小鼠中性粒细胞。这些嗜中性粒细胞没有被激活，并提供无细胞毒性的控制用于在共培养的设置与肿瘤细胞（协议7）肿瘤携带的中性粒细胞。 短半衰期血中性粒细胞与低中性粒细胞数（约3 - 5×10 5）取血1ml的天真6实现- 8周旧鼠标，使人们难以探索小鼠血中性粒细胞功能体外 。中性粒细胞数量稳步在炎症状态和偶尔增加癌症，它代表的慢性炎症7的状态。一些研究人员试图寻找对嗜中性粒细胞的替代来源，如骨髓20。 24小时腹膜内注射1ml的1mg / ml的酵母聚糖A有盐溶液的3％的硫乙醇酸盐肉汤溶液或1毫升后 - 可在4可以得到的大量的小鼠嗜中性粒细胞。但是，这些中性粒细胞引起不发挥ny的抗肿瘤发生活性（未发表的观察）。 。Granot酒店等 6观察到BALB / c小鼠接种原位用小鼠4T1乳腺癌发展嗜中性这恶化时的时间（ 图2A），以使得20 - 4周- 40万血嗜中性粒细胞可从取血1ml 3很容易地分离后肿瘤接种。这些嗜中性粒细胞已获得抗肿瘤活性，并因此被称为肿瘤夹带中性粒细胞（TEN），以便从幼稚中性粒细胞（ 图2B）相区别。而高密度的中性粒细胞（HDN）是高度抗肿瘤发生，在癌症的情况下产生的低密度中性粒细胞（LDN）不是21。从骨髓和荷瘤小鼠的脾高密度的中性粒细胞也具有抗肿瘤活性（未发表数据）。应当指出的是，与肿瘤进展脾脏逐渐放大（脾肿大），以在嗜中性粒细胞压痕量。 为了跟随他们的过继转移追踪中性粒细胞的命运，这些都需要进行标识。嗜中性粒细胞可以通过分离前2天注射溴脱氧尿苷（尿嘧啶）到荷瘤或天真的小鼠被标记在体内 。 BrdU的是该DNA的前体胸苷被掺入新合成的DNA中的增殖细胞的类似物。在嗜中性粒细胞的情况下，BrdU的将并入增殖前体细胞分化为成熟的有丝分裂后的中性粒细胞时保留的BrdU染色。掺入的BrdU可以使用特定的抗BrdU荧光抗体染色。通过流式细胞术对的BrdU +细胞然后可以分析。另一种方法是用细胞追踪染料如5-羧基N-琥珀酰亚胺酯（CFSE）标记分离的嗜中性粒细胞。 CFSE是能够通过活细胞膜的酯化合物。它有一个氨基反应性succinimid这导致共价荧光素结合到在细胞与细胞表面蛋白质和其他氨基基团。的CFSE标记的细胞可以通过流式细胞术使用的488nm氩激光进行分析。两种标记技术的区别在于，BrdU标记依赖于增殖前体细胞，并且不是所有的循环嗜中性粒细胞将被标记，而CFSE会沾污所有嗜中性粒细胞的事实。检测CFSE是不是染色的BrdU更直截了当，但是BrdU标记是跟随不成熟到成熟的中性粒细胞转化的良好手段。 我们还描述了几种方法，以确定嗜中性粒细胞的抗肿瘤和抗转移作用。这些措施包括中性粒细胞耗竭，中性粒细胞过继转移，肿瘤中和试验和肺转移播种法。每个这些测定的完成的抗肿瘤嗜中性粒细胞的功能的特定方面。例如，Granot酒店等 6观察到，当出发嗜中性粒细胞的letion，在4T1肿瘤的小鼠肺转移的数目增加，这表明对于嗜中性粒细胞的抗转移作用。在中性粒细胞过继转移，肿瘤细胞注射纯化HDN前静脉注射4小时。肿瘤细胞形成肺和肝脏转移的能力都遵循一个时间过程研究使用光学体内成像系统。接收HDN小鼠表现出更少的转移灶比没有对照组6。在肿瘤中和试验中，肿瘤细胞被皮下注射或不HDN，HDN的存在降低肿瘤生长21。在转移性播种测定中，GFP标记的肿瘤细胞静脉注射到控制或嗜中性粒细胞的贫预转移性肿瘤的小鼠，并且GFP标记的细胞在肺中的种子的能力是确定的。预转移性肿瘤的小鼠的肺中的特征在于高的中性粒细胞浸润，防止肿瘤细胞接种在特定器官6。这转化为更多转移灶与对照荷瘤小鼠相比嗜中性粒细胞耗尽的小鼠。 该协议描述于研究中性粒细胞功能中的癌症的上下文和提供策略，以评估在体外和体内癌症相关的嗜中性粒细胞特性的焦点。然而，在嗜中性粒细胞纯化策略以及一些所述的实验程序可用于在大范围的实验设置，其中的中性粒细胞发挥 ​​至关重要的作用（ 即，炎症和感染）研究嗜中性粒细胞的功能。

嗜中性粒细胞被初步定性为它作为对抗入侵微生物的第一道防线的先天免疫细胞。目前已知有中性粒细胞更为深远的功能，被卷入安装适应性免疫反应对抗外来抗原1,2，调节造血3，4血管新生和伤口愈合5。此外，嗜中性粒细胞可影响肿瘤的生长和转移进程凭借自己亲和抗肿瘤活性6,7。嗜中性粒细胞的特征是多态分段核（因此称为多形核（PMN）白细胞）和含有颗粒的至少三个不同的亚类，以及分泌囊泡8（ 图1A-C）。 嗜中性粒细胞具有较高的吞噬能力和高NADPH氧化酶的活性微生物消灭批判，并分泌一系列趋化因子在重要的牵引的附加 ​​中性粒细胞和其它免疫细胞到炎症8,9的部位。嗜中性粒细胞的特征是大量的表面受体，包括Toll样受体（TLR）的表达，C型凝集素受体 ​​（的CLR），补体受体3（细胞CD11b / CD18）和其它粘附分子（ 例如，L-选择LFA-1，VLA-4和癌胚抗原相关细胞粘附分子3（CEACAM3 / CD66b）），趋化因子受体（ 例如，CXCR1，CXCR2，CCR1，CCR2），趋化因子受体（ 例如，PAFR，LTB 4 R和C5aR的） ，细胞因子受体（ 例如，G-CSFR，IL-1R，IL-4R，IL-12R，IL-18R，TNFR），甲酰基肽受体（ 例如，FPR1-3），和Fc受体（ 例如，CD16（FcγRIII ），CD32（FcγRII），CD64和（FcγRI）10。在小鼠中，中性粒细胞通常被认定为细胞CD11b + Ly6G +，而人类嗜中性粒细胞使用的CD11b，CD15，CD16和CD66b白细胞识别标志。它也被普遍接受染色的颗粒蛋白髓过氧化物酶（MPO）和中性粒细胞弹性蛋白酶（NE）检测组织中嗜中性粒细胞。 目前还不清楚是否嗜中性粒细胞的不同功能由同一小区或由不同的细胞亚群介导的。累积的数据表明了异种嗜中性白细胞群体表现出可塑性受促炎性刺激物和微环境11,12的高度的存在。 Fridlender 等人 13已经严重分割在癌症中的嗜中性粒细胞分成两个主要的亚群称为N1具有抗肿瘤性质和N2与亲肿瘤性质。在癌症中，以及在慢性炎症中，有一个附加的子种群粒髓源抑制细胞（G-肌源性干细胞），该抑制T细胞反应14组成。 G-肌源性干细胞被认为是其特征在于由一个细胞CD11b +的Ly6C未成熟骨髓细胞低 Ly6G 喜表型的小鼠15，而具有CD15 + / CD16 低人体16的表型。 G-肌源性干细胞表达更高水平的精氨酸酶和髓过氧化物酶，细胞因子和趋化因子比正常循环中性粒细胞，而较低的水平。他们不太吞噬和游走性，但产生较高水平的ROS 15,17,18的。在本文中，我们将介绍一些基本的方法进行隔离中性粒细胞具有抗肿瘤特性和表征。 而中性粒细胞构成的所有白血细胞在人体循环的人口最多（45 - 70％; 1800 - 6000 /微升），在小鼠体内，正常情况下，他们是相当稀疏（10 - 15％; 300 - 500 /微升）。嗜中性粒细胞计数增加时炎症稳步偶尔在癌症，它代表的慢性炎症7的状态。嗜中性粒细胞多能，从普通的骨髓前体（CMP）CE开发LLS在骨髓，通过分化过程通过原粒细胞的阶段（MB），早幼粒细胞（PM），髓细胞（MC），晚幼（MM）和带细胞（BC）8。成熟，有丝分裂后的中性粒细胞可以保持骨髓4内- 7日他们被释放到循环8之前。嗜中性粒细胞周转在血液通常是迅速，平均半衰期为6 - 12小时，这可能是炎症条件下延长。未刺激的嗜中性粒细胞已通过暴露于佛波醇酯佛波12不限抗肿瘤发生活性，可以通过使幼稚中性粒细胞的趋化因子IL-8（CXCL2），CCL2，CCL5和CXCL5 6,19或人工地获取的特征， -myristate 13-乙酸酯（PMA）6。 短半衰期血中性粒细胞与低中性粒细胞数（约3 - 5×10 5）取血1ml的天真6实现- 8周旧鼠标，使人们艰难的探索循环中性粒细胞小鼠体外的功能。为了克服这个困难，其他来源的已被使用。例如，大量的中性粒细胞可以从骨髓20或以下无菌炎症的诱导腹膜得到的（ 例如 ，腹膜内注射硫乙醇酸盐肉汤或酵母聚糖A的之后）。应当指出的是，从腹腔得到嗜中性粒细胞不施加任何抗肿瘤发生活性（未发表的观察）。 。Granot酒店等 6观察到BALB / c小鼠接种原位用小鼠4T1乳腺癌细胞系开发嗜中性这恶化了与肿瘤进展6（ 图2A），以使得20 - 40万血嗜中性粒细胞可以容易地从1毫升血液中分离3 - 第4周后，肿瘤接种。这些嗜中性粒细胞已获得抗肿瘤活性，并因此被瑰奈德肿瘤夹带中性粒细胞（TEN），以便从幼稚中性粒细胞6（ 图2B）相区别。而高密度的中性粒细胞（HDN， 图1A）是高度抗肿瘤发生，在癌症的情况下产生的低密度中性粒细胞（LDN， 图1B）不是21。另外，来自肿瘤携带小鼠的骨髓及脾脏高密度的中性粒细胞具有抗肿瘤活性（未发表数据）。应当指出的是，与肿瘤进展脾脏逐渐放大（脾肿大），以增加嗜中性粒细胞的数量。 应当指出的是，也被在癌症包括自发（MMTV-PyMT和MMTV-WNT1乳腺肿瘤和K-ras基因驱动的肺肿瘤）其他模型生成并注射（AT-3（MMTV-PyMT）和E0771乳腺癌TEN细胞，LLC Lewis肺癌细胞和B16-F10黑素瘤细胞）。然而，在这些TUM中性粒细胞动员的程度或模型远比4T1-接种小鼠的少，达到5 - 10×10 6中性粒细胞在1ml血后3周。

<table><tbody><tr><td>CELL LINES</td><td></td><td></td><td></td></tr><tr><td>Mouse 4T1 breast carcinoma cells</td><td>ADCC</td><td>CRL-2539</td><td>Growth medium: DMEM + 10 % heat-inactivated FBS</td></tr><tr><td>PLASTIC WARES AND EQUIPMENTS</td><td></td><td></td><td></td></tr><tr><td>24-well Tissue Culture Plate&amp;nbsp;</td><td>Falcon</td><td>353047</td><td>Sterile</td></tr><tr><td>100 mm Tissue Culture Plate&amp;nbsp;</td><td>Corning</td><td>430167</td><td>Sterile</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask</td><td>Nunc</td><td>156340</td><td>Sterile</td></tr><tr><td>90 mm Bacterial Grade Culture Dish&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>20090-01-017</td><td>Sterile</td></tr><tr><td>15 ml Sterile Conical Centrifuge Tube&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>835015-40-111</td><td>Sterile</td></tr><tr><td>50 ml Sterile Conical Centrifuge Tube&amp;nbsp;</td><td>Miniplast, Ein Shemer, Israel</td><td>835050-21-111</td><td>Sterile</td></tr><tr><td>Falcon 12 x 75 mm Round-Bottom Polystyrene Tube&amp;nbsp;</td><td>Becton Dickinson</td><td>352058</td><td>Sterile</td></tr><tr><td>Millicell 24 Migration Plate with a pore size of 5&amp;mu;m&amp;nbsp;</td><td>Merck Millipore</td><td>PSMT010R1</td><td>Sterile</td></tr><tr><td>White 96-Flat-Bottom Well Plate&amp;nbsp;</td><td>Costar</td><td>3917</td><td>Sterile</td></tr><tr><td>Cell Strainer (40 mm)&amp;nbsp;</td><td>BD Falcon</td><td>352340</td><td>Sterile</td></tr><tr><td>20G x 1.5&quot; Needle</td><td>BD Microlance 3&amp;nbsp;</td><td>301300</td><td>Sterile</td></tr><tr><td>23G x 1&quot; Needle&amp;nbsp;</td><td>BD Microlance 4</td><td>300800</td><td>Sterile</td></tr><tr><td>25G x 5/8&quot; Needle&amp;nbsp;</td><td>BD Microlance 5</td><td>300600</td><td>Sterile</td></tr><tr><td>0.3 ml Syringe with a 30G x 8mm Needle</td><td>BD Micro-Fine Plus Demi</td><td>320829</td><td>Sterile</td></tr><tr><td>9 mm Clips&amp;nbsp;</td><td>BD, AutoClip&amp;nbsp;</td><td>427631</td><td>Sterile</td></tr><tr><td>EasySep Magnet&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18000</td><td></td></tr><tr><td>MACS LS Separation Column&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-042-201</td><td>Sterile</td></tr><tr><td>MidiMACS Separator Magnet</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Microscope Glass Slide&amp;nbsp;</td><td>Menzel-Gl&amp;auml;ser Superfrost&amp;nbsp; Plus Thermo</td><td>J1800AMNZ</td><td></td></tr><tr><td>Orbital Shaker&amp;nbsp;</td><td>Sky line, ELMI</td><td>S-3.02.10L</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>TECAN</td><td>InfiniteF200Pro</td><td></td></tr><tr><td>POWDER</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA), fraction V</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Bromodeoxyuridine (BrdU)&amp;nbsp;</td><td>BD Pharmingen</td><td>550891</td><td>Sterile</td></tr><tr><td>CFSE (5-(and 6-)-Carboxyfluorescein diacetate, succinimidyl ester)</td><td>Molecular Probes</td><td>C1157</td><td></td></tr><tr><td>Dextran T500</td><td>Sigma</td><td>31392</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa&amp;nbsp;</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S8032</td><td>Highly toxic, handle with care</td></tr><tr><td>Thioglycollate powder&amp;nbsp;</td><td>Difco</td><td>225650</td><td></td></tr><tr><td>Zymosan A</td><td>Sigma</td><td>Z4250</td><td></td></tr><tr><td>MEDIA AND SUPPLEMENTS</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle medium (DMEM)</td><td>Sigma</td><td>D5796</td><td>Sterile</td></tr><tr><td>Opti-MEM&amp;reg; I reduced serum medium&amp;nbsp;</td><td>Life Technologies</td><td>31985062</td><td>Sterile</td></tr><tr><td>Roswell Park Memorial Institute (RPMI)-1640 medium</td><td>Sigma</td><td>R8758</td><td>Sterile</td></tr><tr><td>Foetal bovine serum (FBS), heat-inactivated</td><td>Sigma</td><td>F9665</td><td>Sterile</td></tr><tr><td>L-Glutamine</td><td>Biological Industries, Beth HaEmek, Israel</td><td>03-020-1A</td><td>Sterile</td></tr><tr><td>Sodium pyruvate</td><td>Biological Industries, Beth HaEmek, Israel</td><td>03-042-1B</td><td>Sterile</td></tr><tr><td>Penicillin Streptomycin x 1000 solution</td><td>Biological Industries, Beth HaEmek, Israel</td><td>03-031-5</td><td>Sterile</td></tr><tr><td>Phosphate buffered saline (PBS) without Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-023-1</td><td>Sterile</td></tr><tr><td>PBS x 10 without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-023-5A</td><td>Sterile</td></tr><tr><td>HPLC grade water&amp;nbsp;</td><td>J.T. Baker</td><td>4218-03</td><td>Autoclave</td></tr><tr><td>SOLUTIONS</td><td></td><td></td><td></td></tr><tr><td>ACK &amp;ndash; Ammonium-Chloride-Potassium</td><td>Life Technologies&amp;nbsp;</td><td>A10492-01</td><td></td></tr><tr><td>Bromodeoxyuridine (BrdU) solution (10 mg/ml) in PBS</td><td></td><td></td><td>Dissolve 10 mg of BrdU in 1 ml PBS and sterile filter.</td></tr><tr><td>CFSE, 5 mM in DMSO</td><td></td><td></td><td>Dissolve 2.8 mg of CFSE in 1 ml DMSO. Divide into 10 ml aliquots in sterile 200 ml tubes and store in the dark at -20&lt;sup&gt;o&lt;/sup&gt;C.</td></tr><tr><td>Eosin Y solution</td><td>Sigma</td><td>HT110-2-32</td><td></td></tr><tr><td>Hanks&#039; balanced salt solution</td><td>Biological Industries, Beth HaEmek, Israel</td><td>02-016-1A</td><td>Sterile</td></tr><tr><td>Heparin, 20 mg/ml in PBS</td><td></td><td></td><td>Dissolve 100 mg Heparin in 5 ml sterile PBS, and sterile filter through a 0.2 mm filter.&amp;nbsp;</td></tr><tr><td>Histopaque-1119&amp;nbsp;</td><td>Sigma</td><td>11191</td><td>Sterile filter through a 0.2 mm filter.</td></tr><tr><td>Histopaque-1077&amp;nbsp;</td><td>Sigma</td><td>10771</td><td>Sterile filter through a 0.2 mm filter.</td></tr><tr><td>Luciferase cell culture lysis buffer x5</td><td>Promega</td><td>E153A</td><td>Dilute 1:5 in sterile water just before use.</td></tr><tr><td>Luciferase assay solution</td><td>Promega</td><td>E1501</td><td>Contains luciferase assay substrate powder (E151A) and luciferase assay buffer (E152A)</td></tr><tr><td>Mayer&#039;s Hematoxylin solution&amp;nbsp;</td><td>Sigma</td><td>MHS-32</td><td></td></tr><tr><td>PBS + 0.5% BSA</td><td></td><td></td><td>Dissolve 2.5 g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter.</td></tr><tr><td>PBS + 1% BSA</td><td></td><td></td><td>Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter.</td></tr><tr><td>5x PBS with 2.5% BSA</td><td></td><td></td><td>Dissolve 12.5g BSA in a mixture of 250 ml sterile HPLC-grade water and 250 ml PBSx10, and sterile filter through a 0.2 mm filter.</td></tr><tr><td>PBS containing 0.5% BSA and 2 mM EDTA</td><td></td><td></td><td>Dissolve 250 mg BSA in 50 ml sterile PBS and add 200 ml of 0.5M EDTA pH 8.0, sterile filter through a 0.2 mm filter.</td></tr><tr><td>FACS buffer (PBS containing 0.5% BSA, 2 mM EDTA and 0.02% NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td></td><td></td><td>Dissolve 250 mg BSA in 50 ml sterile PBS&amp;nbsp; and add 200 ml of 0.5M EDTA pH 8.0 and 500 ml of 2% NaN&lt;sub&gt;3&lt;/sub&gt;, sterile filter through a 0.2 mm filter.</td></tr><tr><td>Saline (0.9% NaCl)</td><td></td><td></td><td>Dissolve 9 g NaCl in 1,000 ml ddw, autoclave</td></tr><tr><td>0.2% NaCl solution</td><td></td><td></td><td>Dissolve 2 g NaCl in 1,000 ml ddw, autoclave</td></tr><tr><td>1.6% NaCl solution</td><td></td><td></td><td>Dissolve 16 g NaCl in 1,000 ml ddw, autoclave</td></tr><tr><td>2 % Sodium azide</td><td></td><td></td><td>Dissolve 1g sodium azide in 50 ml sterile ddw, keep at 4&lt;sup&gt;o&lt;/sup&gt;C. Highly toxic.</td></tr><tr><td>3% Thioglycollate solution</td><td></td><td></td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Dissolve 3 g of thioglycollate powder in 100 ml ddw.&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Boil until solution becomes yellow and autoclave.</td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td>Dilute 1:10 in PBS to get a 0.04% solution.</td></tr><tr><td>Trypsin solution B&amp;nbsp;</td><td>Biological Industries, Beth HaEmek, Israel</td><td>03-046-1</td><td>Sterile</td></tr><tr><td>1 mg/ml Zymosan A</td><td></td><td></td><td>Resuspend 1 mg Zymosan A in 1 ml sterile PBS in an Eppendorf tube. Vortex vigorously and incubate the tube at 37 &lt;sup&gt;o&lt;/sup&gt;C for 30 min. Do not autoclave. Prepare the solution freshly before use.</td></tr><tr><td>KITS</td><td></td><td></td><td></td></tr><tr><td>EasySep PE selection kit</td><td>STEMCELL Technologies</td><td>18557</td><td></td></tr><tr><td>EasySep PE selection cocktail&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18151</td><td></td></tr><tr><td>the EasySep magnetic nanoparticles&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18150</td><td></td></tr><tr><td>Anti-Ly6G mouse MicroBead Kit</td><td>Miltenyi Biotec</td><td>130-092-332</td><td></td></tr><tr><td>EasySep Mouse Neutrophil Enrichment Kit</td><td>STEMCELL Technologies</td><td>19762</td><td></td></tr><tr><td>EasySep Human Neutrophil Enrichment Kit</td><td>STEMCELL Technologies</td><td>19257</td><td></td></tr><tr><td>FITC BrdU flow kit</td><td>BD Pharmingen&amp;nbsp;</td><td>559619</td><td></td></tr><tr><td>MACS Neutrophil isolation kit</td><td>Miltenyi Biotec</td><td>130-097-658</td><td></td></tr><tr><td>Phagocytosis Assay Kit&amp;nbsp;</td><td>Cayman Chemical Company&amp;nbsp;</td><td>500290</td><td></td></tr><tr><td>ANTIBODIES</td><td></td><td></td><td></td></tr><tr><td>FcR blocking antibody&amp;nbsp;</td><td>Biolegend</td><td>101302</td><td></td></tr><tr><td>Purified rat anti-Ly6G antibody&amp;nbsp;</td><td>BD Pharmingen&amp;nbsp;</td><td>551459</td><td>Clone 1A8</td></tr><tr><td>PE-conjugated rat anti-mouse Ly6G antibody&amp;nbsp;</td><td>Biolegend</td><td>127608</td><td>Clone 1A8</td></tr><tr><td>FITC-conjugated rat anti-mouse Ly6G</td><td>BD Pharmingen&amp;nbsp;</td><td>551460</td><td>Clone 1A8</td></tr><tr><td>PerCP-Cy5.5 rat anti-mouse Ly6G&amp;nbsp;</td><td>TONBO Biosciences</td><td>65-1276</td><td>Clone 1A8</td></tr><tr><td>violetFluor 450-conjugated rat anti-mouse Ly6G&amp;nbsp;</td><td>TONBO Biosciences</td><td>75-1276</td><td>Clone 1A8</td></tr><tr><td>FITC-conjugated rat anti-mouse CD11b</td><td>BD Pharmingen&amp;nbsp;</td><td>553310</td><td>Clone M1/70</td></tr><tr><td>FITC-conjugated rat anti-mouse Ly-6G and Ly-6C (GR-1)</td><td>BD Pharmingen&amp;nbsp;</td><td>553127</td><td>Clone RB6-8C5</td></tr><tr><td>PE-conjugated rat anti-mouse CD45</td><td>BD Pharmingen&amp;nbsp;</td><td>553081</td><td>Clone 30-F11</td></tr><tr><td>FITC-conjugated rat anti-mouse F4/80&amp;nbsp;</td><td>Abcam</td><td>ab60343</td><td>Clone BM8</td></tr><tr><td>FITC-conjugated mouse anti-human CD66b&amp;nbsp;</td><td>Biolegend</td><td>305103</td><td>Clone G10F5</td></tr><tr><td>Purified rat isotype control antibody (IgG&lt;sub&gt;2a&lt;/sub&gt;, k)&amp;nbsp;</td><td>BD Pharmingen&amp;nbsp;</td><td>553927</td><td>Clone R35-95</td></tr><tr><td>LEAF purified Armenian hamster anti-mouse CD3e antibody</td><td>BioLegend</td><td>100314</td><td>Clone 145-2C11</td></tr></tbody></table>

isolation and characterization of neutrophils with anti-tumor properties

嗜中性粒细胞，最丰富的全白血细胞在人体循环的，起到对入侵微生物的宿主防御中起重要作用。此外，嗜中性粒细胞在肿瘤细胞的免疫监视中心作用​​。他们必须认识到肿瘤细胞，并诱导肿瘤细胞死亡或者通过涉及过氧化氢，或通过抗体依赖性细胞介导的​​细胞毒性（ADCC）的细胞接触依赖性机制的​​能力。嗜中性粒细胞具有抗肿瘤活性可以从癌症患者和荷瘤小鼠的外周血中分离出来。这些嗜中性粒细胞被称为肿瘤夹带的嗜中性粒细胞（TEN）从健康受试者或天真的小鼠表明，没有显著的肿瘤细胞毒活性的嗜中性粒细胞区分开来。与其他的白血细胞相比，嗜中性粒细胞显示出不同的浮力使得当经受密度梯度它可行，得到&gt; 98％纯的嗜中性白细胞群体。然而，除了到正常的高密度嗜中性白细胞群体脱氮（HDN），在癌症患者中，在肿瘤的小鼠，以及下慢性炎性病症，不同的低密度中性粒细胞种群（LDN）出现在循环中。 LDN共纯化的单核组分，并且可以从使用​​正或负选择策略的单核细胞中分离出来。一旦分离嗜中性粒细胞的纯度通过流式细胞术确定的，它们可以被用于在体外和体内功能性测定法。我们描述的技术用于监测中性粒细胞的抗肿瘤活性，它们的迁移，并产生活性氧，以及监测其吞噬能力的离体的能力。我们进一步描述的技术来标记的嗜中性粒细胞在体内跟踪，并确定其在体内的抗转移能力。所有这些技术是必不可少的理解如何获得和具有抗肿瘤表征嗜中性粒细胞功能。

在最近的研究中，我们确定了一个抗转移功能，中性粒细胞6。从荷瘤小鼠嗜中性粒细胞获得细胞毒性表型并具有杀灭肿瘤细胞6的能力。这是相对于那些没有显著抗肿瘤作用6从天真的小鼠嗜中性粒细胞。几个在协议部分中描述的技术已被用于在体外和体内 6研究抗肿瘤嗜中性粒细胞的功能。 肿瘤细胞毒性的中性粒细胞可以从肿瘤携带小鼠6来获得。要达到这个目的，将小鼠原位注射4T1细胞进入左腹股沟乳腺脂肪垫（方案1）。通过后第21天肿瘤接种，原发肿瘤达到1的尺寸- 2 cm 3的（ 图3 -肿瘤是显而易见的，在左下腹部）。此时，对小鼠进行安乐死和1ml抽血（每只小鼠）通过心脏穿刺（ 图3）。与此同时，血液从一个天真的，非荷瘤小鼠被吸引。 HDN然后在密度梯度纯化（协议2.1和图4），得到的中性粒细胞的高纯度（&gt; 98％）的人口。嗜中性粒细胞存活率，台盼蓝染色（协议2.1）确定。的嗜中性粒细胞，然后再悬浮于含有0.5％FBS的2×10 6中性粒细胞/ ml的最优化缩小血清培养基。 为了测试中性粒细胞的细胞毒性的程度，我们加入10 5嗜中性粒细胞（50微升从2×10 6中性粒细胞/ ml储备溶液），以荧光素酶在一个平底白色96表达4T1靶细胞（5000个细胞/孔在100μl） - 嗯板（协议7）。以下的O / N培养，将细胞在PBS中洗涤，并溶解在被动细胞裂解缓冲液和每个样本中的荧光素酶活性进行测试，以便评估中性粒细胞的细胞毒性的程度（ <strong&gt;图2B）（7协议）。荧光素酶的活性与肿瘤细胞存活的数目，并且必须进行比较，以在对照孔中，其中肿瘤细胞是培养的单独的荧光素酶活性。要计算的肿瘤细胞杀伤的程度，我们使用下列公式计算:％的肿瘤细胞杀伤=（1 - 〔中性粒细胞的样品的发光] / [对照样品的发光]）×100％。然后，数据被表示为％细胞不同条件下单独培养的对照细胞具有杀伤（％）= 0。通过使用这些，我们发现，尽管嗜中性粒细胞从无肿瘤小鼠纯化表明对肿瘤细胞无细胞毒性（ 图2B实验程序下杀死的，无肿瘤），嗜中性粒细胞从肿瘤携带小鼠纯化显示显著的细胞毒性（ 图2B，肿瘤轴承）。 为了测试在LDN和加氢脱氮的免疫抑制特性，我们使用了T细胞增殖测定（协议9）。我们评估了CD8 +细胞在未处理的脾细胞，并与一个αCD3抗体处理的脾细胞，这是单独培养，细胞培养在LDN的存在下或在HDN的存在（ 图5A-D）的数量。请注意以下的抗CD3刺激的急剧增加CD8 + CFSE +细胞（比较和右上面板A，B）抑制戏剧性的抑制作用LDN（比较右上面板B和C）以及缺乏HDN的抑制作用（图D）。我们也评估CFSE保留的程度，作为指示对于增殖。在图5E中 ，将橙色曲线呈现未处理CD8 +细胞中，蓝色曲线CD8 +细胞，与αCD3抗体处理，红色曲线CD8 +细胞在LDN和绿色曲线的存在下刺激αCD3代表CD8 +细胞与α刺激; CD3在新生儿溶血病的存在。注意在αCD3处理的细胞中向左移动（蓝色曲线）和αCD3处理的细胞与HDN（绿色曲线）中培养，这表明CD8 +细胞的增殖。 <img alt="图1" src="/files/ftp_upload/52933/52933fig1.jpg" /> 图1.中性粒细胞的形态。高密度（A）和低密度中性粒细胞（B）用苏木和曙红（H＆E）以下的薄层细胞制备的光学显微镜图像。（C）透射电子显微镜（TEM）图像的高密度的中性粒细胞。酒吧代表1000纳米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52933/52933fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52933/52933fig2.jpg" /> 图2.血中性粒细胞计数与肿瘤p增加rogression和中性粒细胞获得细胞毒性表型。 （A）中每毫升的循环嗜中性粒细胞数，通过FACS在以下肿瘤细胞接种在BALB / c小鼠不同天数进行计数。的数量 循环的CD11b + Ly6G +中性粒细胞不断用4T1肿瘤进展而增加。（B）的 4T1乳腺癌细胞共培养以从任天真的小鼠高密度的中性粒细胞（肿瘤免费）或4T1肿瘤的小鼠（肿瘤轴承）小鼠，或培养在介质中不存在嗜中性粒细胞（续）在37℃下20小时。嗜中性粒细胞对肿瘤的小鼠，但不能从肿瘤免费小鼠，表现出对4T1肿瘤细胞显著细胞毒性。误差棒代表±SEM ** P &lt;0.01使用学生t检验。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52933/52933fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alT ="图3"SRC ="/文件/ ftp_upload / 52933 / 52933fig3.jpg"/&gt; 图3.收集的鼠血液通过心脏穿刺 。将小鼠安乐死在感应腔在缓慢的CO 2流。后立即鼠标已采取其末端呼气，它被放置在它的后面和1毫升肝素化注射器插入在胸骨的基部直到到达心脏。柱塞慢慢拉吸出血液。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52933/52933fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52933/52933fig4.jpg" /> 图从全血中性粒细胞4.净化。 （A）3毫升1.077克/ ml的蔗糖小心地层叠在3毫升1.119克/ ml的蔗糖顶部上以形成一个不连续梯度。全鼠血，稀释在PBS-BSA（0.5％），以6 ml的终体积，然后分层对1.07的顶为7g / ml的蔗糖（B）经过30分钟旋转700 XG，没有突破，3个不同的部分，可以观察到。的R -红细胞沉淀中，G -含有高密度的中性粒细胞，M中的粒级分-单核组分含有单核细胞和低密度中性粒细胞（C）的新鲜抽取的人血液与葡聚糖500（等体积混合3％）和在室温下孵育30分钟。含有白血细胞（血沉棕黄层）的顶部馏分，然后层叠在以10ml1.077克/ ml的蔗糖（D）按照30分钟旋在400×g离心，并且没有断点，2个不同的级分，可以观察到顶部。 R + G -含有红细胞和高密度的中性粒细胞，男沉淀-含有单核细胞和低密度单核嗜中性粒细胞比例<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52933/52933fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图5" src="/files/ftp_upload/52933/52933fig5.jpg" /> 图5.抑制的T细胞增殖。流式细胞仪分析表示CFSE标记CD8 +细胞在没有刺激（A）的培养的数量，刺激后αCD3抗体在不存在（B）或LDN的存在（C）的或HDN（D）CFSE强度CD8 +细胞板A的（E）直方图呈现- 。ð <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52933/52933fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Characterization of Neutrophils with Anti-Tumor Properties at JoVE.com

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

isolation-characterization-neutrophils-with-anti-tumor

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

24.7K 次观看.  Hebrew University Medical School. Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo. 

视频: Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

科研

免疫与感染

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

癌症研究

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

将操纵的肿瘤相关中性粒细胞转移到肿瘤携带小鼠中,以研究其血管生成潜力在Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

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.

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 ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

从全血和巴菲皮脂中分离出人类嗜中性粒细胞

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Source: Sionov, R. V., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52933">Isolation and Characterization of Neutrophils with Anti-Tumor Properties.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

从荷瘤小鼠血液中分离细胞毒性中性粒细胞

Source: Sionov, R. V., et al., Isolation and Characterization of Neutrophils with Anti-Tumor Properties. J. Vis. Exp. (2015)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Generating Anti-Angiogenic Tumor-Associated Neutrophils

For tumor-associated neutrophil isolation, place five tumors per well into individual wells of a sterile six-well plate, as they are harvested, and use sterile scissors to mince the tumors into 2- to 3-millimeter pieces. Next, digest the tumor fragments with 1 milliliter of disk-based collagenase, DNase I solution for 45 minutes at 37 degrees Celsius and 5% carbon dioxide with humidity, mixing the samples with a 10-milliliter syringe every 15 minutes.
At the end of the incubation, filter the cells through 100-micrometer strainers into one 15-milliliter tube per well to remove the undigested fibers, and add PBS to a final volume of 15 milliliters. Collect the dissociated cells by centrifugation, and lyse the red blood cells with 1 milliliter of lysis buffer per tube.
After mixing, combine the contents from each tube into a single 15-milliliter tube, stopping the reaction after two minutes with 11 milliliters of complete 4 degrees Celsius medium. After centrifugation, resuspend the pellet in 1 milliliter of PBBS, and block any nonspecific binding with three microliters of FC-blocked antibody for 15 minutes on ice.
At the end of the incubation, label the cells with anti-CD11b and Ly6G antibodies and any other antibodies of interest plus DAPI for a 30-minute incubation on ice protected from light. At the end of the incubation, wash the cells in 14 milliliters of PBS, and resuspend the pellets at a 1 x 107 cells per milliliter of fresh complete medium concentration on ice.
Then, sort the CD11b-positive Ly6G-high DAPI-negative neutrophils on a fluorescence-activated cell sorter according to standard sorting protocols, checking the purity of the isolated neutrophils on the flow cytometer at the end of the sort. For in vitro tumor-associated, neutrophil inhibition, seed 1.5 times 10 to the fifth sorted neutrophils into each of two wells of a 96-well U-bottom plate, and add FK866 to a final concentration of 100 nanomolar in complete medium into one well and an equal volume of complete medium alone to the other well. After two hours in the cell culture incubator, wash the cells two times with PBS, and resuspend the pellets in PBS at the appropriate concentration for the subsequent injection.