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Isolation of Whole Mononuclear Cells from Peripheral Blood
从外周血液中分离全单核细胞   

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本实验方案简略版
Nature Medicine
Feb 2013

Abstract

Whole mononuclear cells from peripheral blood are an easy to obtain and useful population of cells where protein and expression patterns of genes can be studied in patients.

Material and Reagents

  1. EDTA blood (extracted blood kept in tubes with EDTA for anticoagulant purposes)
  2. PBS
  3. Ficoll-Hypaque solution (density 1.077 g/L, Ficoll-Paque PLUS) (General Electric Company, catalog number: 17-1440-03 )
  4. FBS
  5. Complete RPMI-10 (RPMI, 10% FBS)

Equipment

  1. 15- or 50 ml conical centrifuge tube
  2. Beckman GPR centrifuge with GH-3.7 horizontal rotor (or equivalent temperature controlled centrifuge)

Procedure

  1. Place fresh EDTA blood into 15- or 50 ml conical centrifuge tube. Using a sterile pipet, add an equal volume of room temperature PBS. Mix well.
  2. Set a layer of the Ficoll-Hypaque solution at the bottom of the centrifuge tube. Use 3 ml Ficoll-Hypaque per 10 ml blood/PBS mixture.
  3. Slowly layer the blood/PBS over the Ficoll at room temperature. Pipette very slowly the mixture against the wall of the tube so that it does not mix with the Ficoll layer.
  4. Centrifuge 30 min in a GH-3.7 rotor at 900 x g at room temperature, with NO brake.
  5. With a pipette remove the upper layer that contains the plasma and most of the platelets. Transfer the aggregates of mononuclear cells to another centrifuge tube (the intermediate layer contains aggregates of cells presenting white colour and floating over the Ficoll).
  6. Wash the mononuclear cells three times with PBS. Add three times the volume of mononuclear cell layer of PBS. Centrifuge 10 min at 400 x g at room temperature.
  7. Resuspend cells in complete RPMI-10 (RPMI, 10% FBS) (10 ml for plating or 1 ml 10% DMSO for freezing). Freeze or plate the cells.

Acknowledgments

This study was funded by grants SAF2010-17555, RD06/0014/0038 (RECAVA) and, RD06/0010/1013 (TERCEL) from the Spanish Ministry of Economy and Competition (MINECO) and EU FP7-ITN 215761 (NotchIT) to J.L.d.l.P. G.L. had a PhD fellowship from the MINECO (FPI Program, BES-2008-002904).

References

  1. John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, and Warren Strober (eds.) (2004). Curr Protocols Immunol. John Wiley & Sons, Inc. 
  2. Luxan, G., Casanova, J. C., Martinez-Poveda, B., Prados, B., D'Amato, G., MacGrogan, D., Gonzalez-Rajal, A., Dobarro, D., Torroja, C., Martinez, F., Izquierdo-Garcia, J. L., Fernandez-Friera, L., Sabater-Molina, M., Kong, Y. Y., Pizarro, G., Ibanez, B., Medrano, C., Garcia-Pavia, P., Gimeno, J. R., Monserrat, L., Jimenez-Borreguero, L. J. and de la Pompa, J. L. (2013). Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy. Nat Med 19(2): 193-201.

简介

来自外周血的全部单核细胞是容易获得和有用的细胞群,其中可以在患者中研究基因的蛋白质和表达模式。

材料和试剂

  1. EDTA血液(提取的血液保存在用于抗凝血剂目的的EDTA管中)
  2. PBS
  3. Ficoll-Hypaque溶液(密度1.077g/L,Ficoll-Paque PLUS)(General Electric Company,目录号:17-1440-03)
  4. FBS
  5. 完成RPMI-10(RPMI,10%FBS)

设备

  1. 15或50ml圆锥形离心管
  2. 带有GH-3.7水平转子(或同等温度控制的离心机)的Beckman GPR离心机

程序

  1. 将新鲜的EDTA血液置于15或50ml锥形离心管中。 使用无菌移液管,加入等体积的室温PBS。 混合良好。
  2. 在离心管的底部设置一层Ficoll-Hypaque溶液。每10ml血液/PBS混合物使用3ml Ficoll-Hypaque
  3. 在室温下将血液/PBS缓慢地层积在Ficoll上。吸移非常缓慢的混合物对管壁,使其不与Ficoll层混合。
  4. 在室温下在GH-3.7转子中以900×g离心30分钟,用NO制动。
  5. 用移液管移除含有血浆和大多数血小板的上层。将单核细胞的聚集体转移到另一个离心管(中间层含有呈现白色的细胞聚集体,并漂浮在Ficoll上)。
  6. 用PBS洗涤单核细胞三次。加入三倍体积的PBS的单核细胞层。在室温下以400×g离心10分钟。
  7. 重悬细胞在完全RPMI-10(RPMI,10%FBS)(10毫升电镀或1毫升10%的DMSO冻结)。冻结或平板细胞。

致谢

该研究由来自西班牙经济和竞争部(MINECO)的授权SAF2010-17555,RD06/0014/0038(RECAVA)和RD06/0010/1013(TERCEL)和来自J.L.d.l.P.的EU FP7-ITN215761(NotchIT) G.L.有来自MINECO的博士研究生奖学金(FPI计划,BES-2008-002904)。

参考文献

  1. John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M. Shevach和Warren Strober(eds。)(2004)。 Curr Protocols Immunol 。 John Wiley& Sons,Inc. 
  2. Luxan,G.,Casanova,JC,Martinez-Poveda,B.,Prados,B.,D'Amato,G.,MacGrogan,D.,Gonzalez-Rajal,A.Dobarro,D.,Torroja, Martinez,F.,Izquierdo-Garcia,JL,Fernandez-Friera,L.,Sabater-Molina,M.,Kong,YY,Pizarro,G.,Ibanez,B.,Medrano,C.,Garcia-Pavia, ,Gimeno,JR,Monserrat,L.,Jimenez-Borreguero,LJand de la Pompa,JL(2013)。 NOTCH途径调节剂 MIB1 中的突变导致左心室非压缩性心肌病。 Nat Med 19(2):193-201。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martínez-Poveda, B., Luxán, G. and Pompa, J. L. (2013). Isolation of Whole Mononuclear Cells from Peripheral Blood. Bio-protocol 3(20): e939. DOI: 10.21769/BioProtoc.939.
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