In vitro Culture of Human PBMCs
人外周血单核细胞（PBMC）的体外培养
发布: 2013年02月05日第3卷第3期 DOI: 10.21769/BioProtoc.322 浏览次数: 115064
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参见作者原研究论文
The authors used this protocol in:
May 2012
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Abstract
Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.
Materials and Reagents
- Freshly collected heparinised blood
- Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771 )
- Sterile PBS or Dulbecco's modified eagle medium (DMEM) (catalog number: P-04-03590 )
- Pencillin-streptomycin solution (Sigma- Aldrich, catalog number: P4333 )
- W. B. C. diluting fluid (Qualigen, catalog number: 42425 )
- Fetal bovine serum (Pan Biotech, catalog number: 1302-P100402 )
- Trypan blue (Mediatech, catalog number: 193 )
- DMEM supplemented with 1% of Pencillin-streptomycin solution and 10% FBS (see Recipes)
Equipment
- Centrifuge machine with swing-out bucket rotors (Eppendorf, catalog number: 5810 R )
- Heparin vials (BD Biosciences, catalog number: 367886 )
- Sterile 15 ml centrifuge tube
- Auto pipettes
- 200 µl and 1 ml tips
- 24 well cell culture plate (TPP Techno Plastic Products, catalog number: 20090123 )
- Haemocytometer
- Tissue culture hood
- CO2 incubator
- Microscope
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Panda, S. K. and Ravindran, B. (2013). In vitro Culture of Human PBMCs. Bio-protocol 3(3): e322. DOI: 10.21769/BioProtoc.322.
- Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V. and Ravindran, B. (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
细胞生物学 > 细胞分离和培养 > 细胞分离
免疫学 > 免疫细胞分离 > 维持和分化
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