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Isolation of Human PBMCs
人外周血单核细胞(PBMC)的分离   

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Abstract

Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque.

Keywords: PBMC(外周血单个核细胞), Human immune cells(人体免疫细胞), In vitro Culture(体外培养), Monocytes(单核细胞), Lymphocytes(淋巴细胞)

Materials and Reagents

  1. Freshly collected heparinised blood
  2. Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771 ; MP Biomedicals, catalog number: 091692254 )
  3. Sterile PBS or Dulbecco's modified eagle medium (catalog number: P-04-03590)
  4. Pencillin-streptomycin solution (Sigma- Aldrich, catalog number: P-4333 )
  5. Heparin vials (BD Biosciences, catalog number: 367886 )
  6. Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution (see Recipes)

Equipment

  1. Centrifuge machine with swing-out bucket rotor (Eppendorf 5810 R )
  2. Sterile 15 ml centrifuge tube
  3. Auto pipettes and 1 ml tips

Procedure

  1. Collect 4 ml of human venous blood sample in heparinised vials (BD biosciences) and mix well by gently inverting the tube several times.
  2. Take 4 ml of Ficoll Histopaque in a 15 ml centrifuge tube.
  3. Gently layer the blood on the top of Ficoll Histopaque using a 1 ml auto pipette. The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers.
  4. Centrifuge the tubes (without any delay) for 30 min at 100 x g in 4 °C in a swing-out bucket. Fixed angle rotors also can be used but would require more caution when separating cells in interphase.
  5. Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium.
  6. The cells in interphase need to be aspirated without delay. If the tubes are kept standing for more than 10 min, PBMCs from the interphase will get disturbed and start settling down.
  7. Wash (centrifuge in 100 x g for 10 min) twice with 10 ml of sterile PBS or sterile Dulbecco's modified eagle medium. The approximate yield of cells from 4 ml of blood varies between 107-108.

Recipes

  1. Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution

Technical notes

  1. Use freshly collected heparinised blood. If plasma needs to be used for any other purpose, then remove the plasma and add equal volume of sterile Dulbecco's modified eagle medium before proceeding further.
  2. The ratio between Ficoll Histopaque and blood should be 1:1 for human blood. The ratio could be different for purification of PBMC of other species.
  3. Ficoll Histopaque is stored at 4 °C. Before use the tube needs to be kept at room temperature for 1-2 h since PBMCs will get cold shock or sometimes aggregate if layered in pre-chilled Ficoll Histopaqueon blood sample.

Acknowledgments

The laboratory protocol was evolved over time in the senior authors’ laboratory using a template that was published in 1986 in Handbook of Experimental Immunology / edited by D. M. Weir; co-editors, L. A. Herzenberg, Caroline Blackwell, Leonore A. Herzenberg. Blackwell Scientific Publications. Institute of Life Sciences is funded by Department of Biotechnology, Govt of India and SP was supported by a fellowship grant from Indian Council of Medical Research.

References

  1. Panda, S. K., S. Kumar, N. C. Tupperwar, T. Vaidya, A. George, S. Rath, V. Bal and B. Ravindran (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.

简介

外周血单核细胞(PBMC)主要是淋巴细胞和单核细胞。 通过使用Ficoll Histopaque的密度梯度离心法从全血中分离PBMC。

关键字:外周血单个核细胞, 人体免疫细胞, 体外培养, 单核细胞, 淋巴细胞

材料和试剂

  1. 新鲜收集的肝素化血液
  2. Ficoll Histopaque(Sigma-Aldrich,目录号:10771; MP Biomedicals,目录号:091692254)
  3. 无菌PBS或Dulbecco's改良的Eagle培养基(目录号:P-04-03590)
  4. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P-4333)
  5. 肝素小瓶(BD Biosciences,目录号:367886)
  6. 补充有1%青霉素 - 链霉素溶液(参见Recipes)的Dulbecco's改良的Eagle培养基

设备

  1. 带离心式转子离心机(Eppendorf 5810 R)
  2. 无菌15ml离心管
  3. 自动移液器和1 ml提示

程序

  1. 在肝素化小瓶(BD biosciences)中收集4ml人静脉血样品,并轻轻颠倒该管几次混合均匀。
  2. 取4 ml Ficoll Histopaque在15 ml离心管中
  3. 使用1毫升自动移液器轻轻地分层的血液在Ficoll Histopaque的顶部。 分层应该非常缓慢,血液和Ficoll Histopaque应该保持为两个不同的层
  4. 在4℃下在摇摆桶中以100×g离心管(无任何延迟)30分钟。 也可以使用定角转子,但是在分离期间分离细胞时需要更加小心
  5. 吸出在间质细胞和培养基之间形成的白色血沉棕黄层(约1ml)(PBMC)。
  6. 相间的细胞需要立即被吸出。 如果管保持静置超过10分钟,来自间期的PBMC将被扰乱并开始沉降。
  7. 用10ml无菌PBS或无菌Dulbecco's改性Eagle培养基洗涤(用100×g离心10分钟)两次。 来自4ml血液的细胞的近似产率在10 -10 8 之间变化。

食谱

  1. Dulbecco's改良的Eagle培养基,补充有1%的青霉素 - 链霉素溶液

技术说明

  1. 使用新鲜收集的肝素化血液。如果等离子体需要用于任何其他目的,则在进一步处理之前,除去血浆并加入等体积的无菌Dulbecco's改良的Eagle培养基。
  2. 对于人类血液,Ficoll Histopaque和血液之间的比例应该为1:1。该比例对于其它物种的PBMC的纯化可以是不同的
  3. Ficoll Histopaque在4℃储存。使用前,管需要保持在室温下1-2小时,因为PBMC会得到冷休克或有时聚集,如果在预冷的Ficoll Histopaqueon血液样本中。

致谢

实验室方案在高级作者实验室中使用1986年在Handbook of Experimental Immunology/edited by D.M.Weir中发表的模板随时间演变;共同编辑,L. A. Herzenberg,Caroline Blackwell,Leonore A. Herzenberg。 Blackwell科学出版社。生命科学研究所由生物技术部资助,印度政府,SP得到印度医学研究委员会的奖学金资助。

参考文献

  1. Panda,S.K.,S.Kumar,N.C.Tupperwar,T.Vaidya,A.Jorge,S.Rath,V.Bal和B.Ravindran(2012)。 Chitohexaose通过TLR4的交替途径激活巨噬细胞,阻断内毒素血症。 PLoS Pathog 8(5):e1002717。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Panda, S. K. and Ravindran, B. (2013). Isolation of Human PBMCs. Bio-protocol 3(3): e323. DOI: 10.21769/BioProtoc.323.
  2. Panda, S. K., S. Kumar, N. C. Tupperwar, T. Vaidya, A. George, S. Rath, V. Bal and B. Ravindran (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.
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