Monocyte-MSC Co-cultures
单核细胞-MSC 共培养物
发布: 2015年01月20日第5卷第2期 DOI: 10.21769/BioProtoc.1384 浏览次数: 11744
评审: Hui ZhuNingfei AnAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2013
Abstract
To assess the effect of multipotent stromal cells (MSC) on monocytes, 3-day cultures were performed of freshly isolated monocytes in MSC-conditioned medium (CM). As a control condition, monocytes were stimulated with low dose macrophage colony-stimulating factor (M-CSF). Monocytes were isolated from peripheral blood mononuclear cell (PBMC) populations by magnetic activated cell sorting (MACS) using CD14 microbeads.
Materials and Reagents
- Peripheral blood mononuclear cells (PBMC) [isolated from a buffy coat from a healthy donor using Ficoll-Paque (own pharmacy) density gradient (1.077 g/cm3)]
- Multipotent stromal cells (MSC) from healthy donors
- Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
- Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
- L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
- Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758072 )
- Phosphate buffered saline (PBS) (produced by in-house pharmacy)
- M-CSF (Pepro Tech, catalog number: 300-25 )
- CD14 MicroBeads (human) (Miltenyi Biotec, catalog number 130-050-201 )
- Antibodies
- Anti-CD14 PE (BD Biosciences)
- Anti-CD206 APC (BD Biosciences)
- Anti-CD163 PerCP-Cy5.5 (BioLegend)
- Anti-CD80 PE-Cy7 (BioLegend)
- Anti-CD14 PE (BD Biosciences)
- Trypsin/EDTA (0.05%, phenol red) (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
- Culture medium (see Recipes)
- Proliferation medium (see Recipes)
Equipment
- T75 culture flasks
- 6-well plates (Sigma-Aldrich, catalog number: CLS3506 )
- 37 °C, 5% CO2 cell culture incubator
- Microscope
- Centrifuge
- Hemocytometer (counting chamber)
- MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
- MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
- MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
- 24-well plates (Sigma-Aldrich, catalog number: CLS3526 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Melief, S. M., Schrama, C. L. M. and Roelofs, H. (2015). Monocyte-MSC Co-cultures. Bio-protocol 5(2): e1384. DOI: 10.21769/BioProtoc.1384.
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分类
干细胞 > 成体干细胞 > 造血干细胞
干细胞 > 成体干细胞 > 基质细胞
干细胞 > 成体干细胞 > 维持和分化
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