Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol
采用简单的逆流层析法纯化人单核细胞和淋巴细胞群
发布: 2013年12月05日第3卷第23期 DOI: 10.21769/BioProtoc.981 浏览次数: 11584
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参见作者原研究论文
The authors used this protocol in:
Jun 1986
Abstract
Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been shown to be appropriately capable of differential activation in multiple studies.
Keywords: Monocyte (单核细胞)
Human (人类)
Isolation (隔离)
Elutriation (淘洗)
Lymphocyte (淋巴细胞)
Materials and Reagents
- Peripheral blood from normal human donor (450 ml)
- CPD AS-1 blood collection bags (Citrate-Phosphate-Dextrose, Adenine saline-1) (Baxter)
- 1x PBS-2mM EDTA
- Sterile milliQ H2O
- Lymphocyte separating medium (LSM) (MP Biomedicals, catalog number: 0 850494 )
- HBSS (Fisher Scientific, catalog number: 21023CV )
- Ethanol (70%, 1 liter)
- 10% Bleach (1 liter)
- 10% defined FBS (Hyclone, catalog number: SH30070.03 )
- 1% P/S (Mediatech, CellGro®, catalog number: 30-002-CIRF )
- 1% L-Gln (Mediatech, CellGro®, catalog number: 25-005-CI )
- Recombinant human IL-2 (Pepro Tech, catalog number: 200-02 )
- Recombinant human M-CSF (Pepro Tech, catalog number: 300-25 )
- 25% Albumin (Human), USP (Talecris Plasma Resources, catalog number: 13533-684-20 )
- Complete media (see Recipes)
- 1x sterile PBS (dilute 1:5 from 5x PBS pH 7.0 – 7.2) (see Recipes)
- Ammonium Chloride Potassium (ACK) buffer (see Recipes)
- 1x sterile Elutriation Buffer (see Recipes)
- 5x Phosphate Buffered Saline (see Recipes)
Equipment
- Extruder
- T75 flasks
- Nunc blue cap 50-ml polypropylene tubes (important as monocytes stick to other tubes lowing yields)
- Tubing clamps
- Scissors
- P1000 Pipetteman
- Petri dishes
- 250 ml plasma tube
- Isopropanol wipes, individually packaged
- Beckman JE6B Elutriation System and Rotor (Beckman Coulter, model: JE6B-IM-3) (November 1992)**
- Centrifuge (Sorvall, model: RT6000 or RC-3B )
- Stericup (Fisher Scientific, catalog number: SCGVU11RE )
** Beckman Coulter has newer models Avanti J-301 or J-26S XP with Elutriation Systems, but we have no experience with those.
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Clarke, E. V., Benoit, M. E. and Tenner, A. J. (2013). Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol. Bio-protocol 3(23): e981. DOI: 10.21769/BioProtoc.981.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
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