小鼠血液中血小板的纯化
Summary
在这里, 老鼠的血液是在抗凝剂的存在下采集的。采用碘己醇梯度介质, 采用低速离心法纯化血小板。血小板被激活与凝血酶调查, 他们是否可行。采用流式细胞仪和显微镜对纯化后的血小板质量进行了分析。
Abstract
血小板从全血中纯化, 以研究其功能特性, 这些功能应从红细胞 (RBC)、白细胞 (WBC) 和血浆蛋白中解脱出来。我们在这里描述纯化血小板从小鼠血液使用三倍以上的 iohexol 梯度介质相对于血液样本量和离心在一个摇摆桶转子在 400 x g在 20°c 20分钟。纯化后的血小板的恢复率为 18.2-38.5%, 纯化后的血小板处于休眠状态, 不含明显的红细胞和 WBC。用凝血酶处理的纯化血小板的活化率达到 93%, 表明其活力。我们证实, 纯化后的血小板是足够纯净的流式细胞仪和微观评价。这些血小板可用于基因表达、活化、颗粒释放、聚集和粘附检测。该方法也可用于从其他物种的血液中纯化血小板。
Introduction
血小板是血液的一个组成部分, 它起到血液凝集剂的作用, 以应对血管的损伤。他们聚集在受伤地点, 堵塞船只墙1号。血小板是由骨髓巨核细胞在血栓生成素的影响下产生的细胞质的无核碎片, 进入循环2。它们被认为是代谢活跃的, 能够通过激活细胞内信号级联来感知细胞外环境, 从而导致血小板扩散、聚集和止血塞形成1,3。除了止血/血栓形成和伤口愈合 4, 血小板在宿主炎症反应, 血管生成和转移3,5,6,7,8。
血小板从血液中纯化, 以研究其生化和生理特性, 这些特性应该从其他血液成分中解脱出来。由于红细胞 (rbc) 和白细胞 (wbc) 的 rna 和蛋白质含量明显高于血小板9,10, 即使是少数这些细胞的存在也会干扰 rna 的转录和蛋白质组学分析和从血小板中提取的蛋白质。我们发现, 用凝血酶激活的纯化血小板比全血小板更有效地结合抗 Gpib/iiia (jonsa) 和抗 p-选择素等抗体。
由于血小板是脆弱的, 重要的是要尽可能温和地对待样品。如果血小板被激活, 它们释放颗粒内的物质, 并最终降解。因此, 为了保持血小板的功能特性完整, 在隔离过程中保持血小板的静止是非常重要的。一些协议描述了通过各种方法1、10、11、12将血小板与人类、狗、老鼠和非人类灵长类动物隔离。其中一些方法需要多个步骤, 如通过离心收集富含血小板的血浆, 通过分离柱过滤, 负选择血小板与 rbc-和 wbc 特定抗体共轭磁珠, 等等, 这些步骤是耗时, 并可能降解血小板及其内容物。
福特和他的同事描述血小板纯化从人类血液使用 iohexol 培养基11。这种方法在纯化过程中使用了类似体积的血液样本和培养基。由于人类产生较高的血液量, 因此相对容易净化血小板。
即异己醇是一种通用密度梯度惰性介质, 可自由溶于水, 用于核酸、蛋白质、多糖和核蛋白13、14的分馏。它具有低渗透性和无毒, 从而成为纯化完整活细胞11 的理想培养基。它是一种非颗粒介质;因此, 细胞在梯度的分布可以用血细胞仪, 流式细胞仪, 或分光光度计来确定。稀释后, 它不会干扰细胞或细胞碎片的大部分酶反应或化学反应。
老鼠是许多人类疾病15、16、17、18的重要动物模型。有发表的一些文章, 描述纯化的小鼠血小板 19,20。然而, 小鼠产生的血液量相对较小, 这使得血小板难以净化。如果使用相同体积的梯度介质和血液样本, 离心后血小板层不能与 RBC-WBC 层清楚分离。本文介绍了一种快速简便的小鼠血小板纯化方法, 该方法的方法是将异丙醇梯度培养基的三倍以上, 相对于血样体积和低速离心。我们还用凝血酶激活了纯化后的血小板, 并用流式细胞仪和显微镜对其质量进行了研究。
Protocol
Representative Results
Discussion
通常情况下, 血小板是通过低速离心分离的, 这种离心法产生富含血小板的血浆, 其中含有大量的血细胞、细胞碎片和血浆蛋白, 这些细胞、细胞碎片和血浆蛋白会干扰生化和生理检测和需求进一步净化21。因此, 重要的是要使用一种快速和简单的方法, 可以产生纯血小板, 而不会产生主要的污染物。这里介绍的协议描述了使用梯度介质和低速离心从小鼠血液中纯化血小板的方法。该…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了辛辛那提儿童研究基金会的启动资金和辛辛那提大学向 m. n. 提供的试点翻译赠款的支持。我们要感谢辛辛那提儿童医院研究流式细胞仪核心公司的服务。
Materials
| APC rat anti-mouse/human CD62P (P-selectin) | Thermoscientific | 17-0626-82 | Platelets activation marker |
| Eppendorf tube | Fisher Scientific | 14-222-166 | Tube for centifuge |
| FACS DIVA software | BD Biosciences | Non-catalog item | Analysis of platelets and whole blood |
| FACS tube | Fisher Scientific | 352008 | Tubes for flow cytomtery |
| Fetal bovine serum (FBS) | Invitrogen | 26140079 | Ingredient for staining buffer |
| FITC rat anti-mouse CD41 | BD Biosciences | 553848 | Platelets marker |
| Flow cytometer | BD Biosciences | Non-catalog item | Analysis of platelets and whole blood |
| FlowJo software | FlowJo, Inc. | Non-catalog item | Analysis of platelets and whole blood |
| Gly-Pro-Arg-Pro (GPRP) | EMD Millipore | 03-34-0001 | Prevent platelet clot formation |
| Hematocrit Capillary tube | Fisher Scientific | 22-362566 | Blood collection capillary tube |
| Hemavet | Drew Scientific | Non-catalog item | Blood cell analyzer |
| Hemocytometer | Hausser Scientific | 3100 | Cell counting chamber |
| Isoflurane | Baxter | 1001936040 | Use to Anesthetize mouse |
| Microscope (Olympus CKX41) | Olympus | Non-catalog item | Cell monitoring and counting |
| Nycodenz (Histodenz) | Sigma-Aldrich | D2158 | Gradient medium |
| PE rat anti-mouse CD45 | BD Biosciences | 561087 | WBC marker |
| PE-Cy7 rat anti-mouse TER 119 | BD Biosciences | 557853 | RBC marker |
| Pipet tips 200 µL, wide-bore | ThermoFisher Scientific | 21-236-1A | Transferring blood and platelet samples |
| Pipet tips 1000 µL, wide-bore | ThermoFisher Scientific | 21-236-2C | Transferring blood and platelet samples |
| Phosphate buffured saline (PBS) | ThermoFisher Scientific | 14040-117 | Buffer for washing and dilution |
| Sodium chloride | Sigma-Aldrich | S7653 | Physiological saline |
| Sodium citrate | Fisher Scientific | 02-688-26 | Anti-coagulant |
| Staining buffer | In-house | Non-catalog item | Wash and dilution buffer |
| Steile water | In-house | Non-catalog item | Solvent |
| Table top centrifuge | ThermoFisher Scientific | 75253839/433607 | Swinging bucket rotor centrifuge |
| Thrombin | Enzyme Research Laboratoty | HT 1002a | Platelet activation agonist |
| Tricine | Sigma-Aldrich | T0377 | Buffer for Nycodenz medium |
References
- de Witt, S. M., Verdoold, R., Cosemans, J. M., Heemskerk, J. W. Insights into platelet-based control of coagulation. Thrombosis Research. 133, S139-S148 (2014).
- Machlus, K. R., Thon, J. N., Italiano, J. E. Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular processes mediating platelet formation. British Journal of Haematology. 165 (2), 227-236 (2014).
- Weyrich, A. S., Zimmerman, G. A. Platelets: signaling cells in the immune continuum. Trends in Immunology. 25 (9), 489-495 (2004).
- Nami, N., et al. Crosstalk between platelets and PBMC: New evidence in wound healing. Platelets. 27 (2), 143-148 (2016).
- Mancuso, M. E., Santagostino, E. Platelets: much more than bricks in a breached wall. British Journal of Haematology. 178 (2), 209-219 (2017).
- Hampton, T. Platelets’ Role in Adaptive Immunity May Contribute to Sepsis and Shock. Journals of the American Medical Association. 319 (13), 1311-1312 (2018).
- Sabrkhany, S., Griffioen, A. W., Oude Egbrink, M. G. The role of blood platelets in tumor angiogenesis. Biochimica et Biophysica Acta (BBA) – Reviews on Cancer. 1815 (2), 189-196 (2011).
- Menter, D. G., et al. Platelet "first responders" in wound response, cancer, and metastasis. Cancer and Metastasis Reviews. 36 (2), 199-213 (2017).
- Fink, L., et al. Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection. Thrombosis and Haemostasis. 90 (4), 749-756 (2003).
- Trichler, S. A., Bulla, S. C., Thomason, J., Lunsford, K. V., Bulla, C. Ultra-pure platelet isolation from canine whole blood. BioMed Central Veterinary Research. 9, 144 (2013).
- Ford, T. C., Graham, J., Rickwood, D. A new, rapid, one-step method for the isolation of platelets from human blood. Clinica Chimica Acta. 192 (2), 115-119 (1990).
- Noisakran, S., et al. Infection of bone marrow cells by dengue virus in vivo. Experimental Hematolology. 40 (3), 250-259 (2012).
- Rickwood, D., Ford, T., Graham, J. Nycodenz: a new nonionic iodinated gradient medium. Analytical Biochemistry. 123 (1), 23-31 (1982).
- Graham, J. M., Ford, T., Rickwood, D. Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge. Analytical Biochemistry. 187 (2), 318-323 (1990).
- Milligan, G. N., et al. A lethal model of disseminated dengue virus type 1 infection in AG129 mice. Journal of General Virology. 98 (10), 2507-2519 (2017).
- Strassel, C., et al. Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibbeta function. Journal of Thrombosis and Haemostasis. 14 (7), 1470-1479 (2016).
- Bergmeier, W., Boulaftali, Y. Adoptive transfer method to study platelet function in mouse models of disease. Thrombosis Research. 133, S3-S5 (2014).
- Bakchoul, T., et al. Recommendations for the use of the non-obese diabetic/severe combined immunodeficiency mouse model in autoimmune and drug-induced thrombocytopenia: communication from the SSC of the ISTH. Journal of Thrombosis and Haemostasis. 13 (5), 872-875 (2015).
- Aurbach, K., Spindler, M., Haining, E. J., Bender, M., Pleines, I. Blood collection, platelet isolation and measurement of platelet count and size in mice-a practical guide. Platelets. , 1-10 (2018).
- Jirouskova, M., Shet, A. S., Johnson, G. J. A guide to murine platelet structure, function, assays, and genetic alterations. Journal of Thrombosis and Haemostasis. 5 (4), 661-669 (2007).
- Birschmann, I., et al. Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway. Thrombosis Research. 122 (1), 59-68 (2008).
