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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, 2194 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


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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