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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.

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Monocyte-MSC Co-cultures
单核细胞-MSC 共培养物

干细胞 > 成体干细胞 > 造血干细胞
作者: Sara M. Melief
Sara M. MeliefAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a1952
C. L. M. Schrama
C. L. M. SchramaAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a1953
 and Helene Roelofs
Helene RoelofsAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
For correspondence: h.roelofs@lumc.nl
Bio-protocol author page: a1954
Vol 5, Iss 2, 1/20/2015, 3269 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1384

[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.

[Abstract]

Materials and reagents

  1. 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)]
  2. Multipotent stromal cells (MSC) from healthy donors
  3. Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
  4. Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
  5. L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
  6. Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758072 )
  7. Phosphate buffered saline (PBS) (produced by in-house pharmacy)
  8. M-CSF (Pepro Tech, catalog number: 300-25 )
  9. CD14 MicroBeads (human) (Miltenyi Biotec, catalog number 130-050-201 )
  10. Antibodies
    1. Anti-CD14 PE (BD Biosciences)
    2. Anti-CD206 APC (BD Biosciences)
    3. Anti-CD163 PerCP-Cy5.5 (BioLegend)
    4. Anti-CD80 PE-Cy7 (BioLegend)
  11. Trypsin/EDTA (0.05%, phenol red) (Life Technologies, InvitrogenTM, catalog number: 25300-054 )
  12. Culture medium (see Recipes)
  13. Proliferation medium (see Recipes)

Equipment

  1. T75 culture flasks
  2. 6-well plates (Sigma-Aldrich, catalog number: CLS3506 )
  3. 37 °C, 5% CO2 cell culture incubator
  4. Microscope
  5. Centrifuge
  6. Hemocytometer (counting chamber)
  7. MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
  8. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  9. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  10. 24-well plates (Sigma-Aldrich, catalog number: CLS3526 )

Procedure

  1. MSC cultures are generated from aspirated bone marrow: bone marrow-derived mononuclear cells are isolated using Ficoll-Paque density gradient (1.077 g/cm3) and plated at 1.3 x 105 cells/cm2 in proliferation medium.
  2. Cultures were incubated at 37 °C and 5% CO2. After 3-4 days non-adherent cells were removed, and medium was refreshed every 3-4 days until confluence was reached. The MSC monolayer was detached using trypsin/EDTA, and cells were reseeded at 4,000 cells per cm2 for further expansion.
  3. MSC (passage 2-5) are cultured at confluency in a T75 culture flasks in 10 ml culture medium for at least 3 days without refreshing the medium.
  4. The medium is aspirated from the cultures (= MSC-CM). Spin down MSC-CM at 350 x g for 10 min to obtain cell-free MSC-CM.
  5. Day 0: Isolate monocytes from freshly obtained PBMC from a buffy coat by MACS using CD14 microbeads and MS Columns according to the manufacturer’s instructions.
  6. Plate 2.5 x 106 monocytes in a 24-wells plate in 400 µl culture medium.
  7. Add 600 µl of cell-free MSC-CM to the monocyte cultures.
  8. As control condition, add M-CSF to the monocyte cultures at a concentration of 5 ng/ml.
  9. Place the cultures for 3 days at 37 °C in a 5% CO2 cell culture incubator.
  10. Day 3: Collect monocytes.
    Note: When the monocytes are attached, place the plates for 15 min on ice.
  11. Spin down monocytes at 350 x g for 10 min.
  12. Add 120 µl of PBS and count dead/alive cells using a hemocytometer.
  13. Analyze monocytes with flowcytometry/ isolate mRNA for follow-up analysis.
    1. Antibodies for flowcytometry to analyze the monocytes:
      1. Anti-CD14 PE
      2. Anti-CD206 APC
      3. Anti-CD163 PerCP-Cy5.5
      4. Anti-CD80 PE-Cy7

Recipes

  1. Culture medium
    RPMI medium
    10% FCS
    P/S (100 U/ml)
    L-glutamin (100 U/ml)
  2. Proliferation medium
    Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG)
    10% FCS
    P/S (100 U/ml)
    L-glutamin (100 U/ml)

References

  1. Melief, S. M., Schrama, E., Brugman, M. H., Tiemessen, M. M., Hoogduijn, M. J., Fibbe, W. E. and Roelofs, H. (2013). Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells 31(9): 1980-1991.

材料和试剂

  1. 外周血单核细胞(PBMC)[使用Ficoll-Paque(自己的药学)密度梯度(1.077g/cm 3 )从健康供体的血沉棕黄层分离]
  2. 来自健康捐献者的多功能基质细胞(MSC)
  3. Roswell Park Memorial Institute(RPMI)1640培养基(Life Technologies,目录号:31870-082)
  4. 青霉素/链霉素(5,000U/ml)(Life Technologies,目录号:15070-063)
  5. L-谷氨酰胺(200mM)(Life Technologies,目录号:25030-024)
  6. 胎牛血清(FCS)(Greiner Bio-One GmbH,目录号:758072)
  7. 磷酸盐缓冲盐水(PBS)(由内部药房生产)
  8. M-CSF(Pepro Tech,目录号:300-25)
  9. CD14 MicroBeads(人)(Miltenyi Biotec,目录号130-050-201)
  10. 抗体
    1. 抗CD14PE(BD Biosciences)
    2. 抗CD206 APC(BD Biosciences)
    3. 抗CD163 PerCP-Cy5.5(BioLegend)
    4. 抗CD80 PE-Cy7(BioLegend)
  11. 胰蛋白酶/EDTA(0.05%,酚红)(Life Technologies,Invitrogen TM,目录号:25300-054)
  12. 培养基(见配方)
  13. 增殖培养基(参见配方)

设备

  1. T75培养瓶
  2. 6孔板(Sigma-Aldrich,目录号:CLS3506)
  3. 37℃,5%CO 2细胞培养箱中培养
  4. 显微镜
  5. 离心机
  6. 血细胞计数器(计数室)
  7. MS柱(Miltenyi Biotec,目录号:130-042-201)
  8. MiniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  9. MACS MultiStand(Miltenyi Biotec,目录号:130-042-303)
  10. 24孔板(Sigma-Aldrich,目录号:CLS3526)

程序

  1. MSC培养物从吸出的骨髓产生:使用Ficoll-Paque密度梯度(1.077g/cm 3)分离骨髓衍生的单核细胞,并以1.3×10 5/>细胞/cm 2在增殖培养基中。
  2. 将培养物在37℃和5%CO 2孵育。 3-4天后,除去非贴壁细胞,每3-4天更新培养基直至达到融合。使用胰蛋白酶/EDTA分离MSC单层,并以4,000个细胞/cm 2再次接种细胞以进一步扩增。
  3. MSC(2-5代)在T75培养瓶中在汇合下在10ml培养基中培养至少3天而不更新培养基。
  4. 从培养物中吸出培养基(= MSC-CM)。在350×g下旋转MSC-CM 10分钟以获得无细胞的MSC-CM。
  5. 第0天:根据制造商的说明书,使用CD14微珠和MS柱,通过MACS从血沉棕黄层中分离来自新鲜获得的PBMC的单核细胞。
  6. 在400μl培养基中的24孔板中铺板2.5×10 6个单核细胞。
  7. 向单核细胞培养物中加入600μl无细胞MSC-CM
  8. 作为对照条件,将M-CSF以5ng/ml的浓度加入单核细胞培养物中
  9. 将培养物在37℃下在5%CO 2细胞培养箱中放置3天。
  10. 第3天:收集单核细胞。
    注意:当连接单核细胞时,将板在冰上放置15分钟。
  11. 在350×g下旋转单核细胞10分钟。
  12. 加入120μlPBS,并使用血细胞计数器计数死/活细胞
  13. 用流式细胞术/分离mRNA分析单核细胞用于随访分析
    1. 用于流式细胞术分析单核细胞的抗体:
      1. 抗CD14 PE
      2. 抗CD206 APC
      3. 抗CD163 PerCP-Cy5.5
      4. 抗CD80 PE-Cy7

食谱

  1. 培养基
    RPMI培养基
    10%FCS
    P/S(100U/ml) L-谷氨酰胺(100U/ml)
  2. 增殖培养基
    Dulbecco改良的Eagle's中低葡萄糖(DMEM-LG)
    10%FCS
    P/S(100U/ml) L-谷氨酰胺(100U/ml)

参考文献

  1. Melief,S.M.,Schrama,E.,Brugman,M.H.,Tiemessen,M.M.,Hoogduijn,M.J.,Fibbe,W.E.and Roelofs,H。(2013)。 多功能基质细胞通过涉及单核细胞向抗炎巨噬细胞倾斜的新途径诱导人类调节性T细胞 。 Stem Cells 31(9):1980-1991。
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How to cite this protocol: Melief, S. M., Schrama, C. L. and Roelofs, H. (2015). Monocyte-MSC Co-cultures. Bio-protocol 5(2): e1384. DOI: 10.21769/BioProtoc.1384; Full Text



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