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Clonal Culture of Mouse Liver Progenitor Cells

小鼠肝脏前体细胞的克隆培养

NT Naoki Tanimizu

发布: 2015年10月20日第5卷第20期 DOI: 10.21769/BioProtoc.1624 浏览次数: 12040

评审: Anonymous reviewer(s)

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Abstract

Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. For analyzing their differentiation potential, clonal culture has been used for LPCs isolated by a cell sorter. In addition, we can use the culture to assess functions of target genes on differentiation potential of LPCs. This protocol describes the process of cell isolation and colony assay to examine proliferative and differentiation potential of LPCs.

Keywords: Bipotential (双电位) search Hepatocyte (肝细胞) search Cholangiocyte (胆管上皮细胞) search Liver (肝脏) search Flow cytometry (流式细胞仪) search

Materials and Reagents

  1. Autoclaved 250 μm Nylon mesh (Nippon Rikagaku Kikai)
  2. Butterfly needle (23 gauge) (Terumo, catalog number: SV-23CLK )
  3. FalconTM Cell Strainers (Falcon, catalog number: 332350 )
    Note: Currently, it is “Thermo Fisher Scientific, Falcon™, catalog number: 332350 ”.
  4. 35 mm tissue culture dish (Corning, catalog number: 430165 )
  5. Collagenase (used at 1 mg/ ml for perfusion) (Wako Pure Chemical Industries, catalog number: 032-10534 )
  6. PBS
  7. Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9269 )
  8. Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (Sigma-Aldrich, catalog number: E0396 )
  9. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  10. Hyaluronidase (Sigma-Aldrich, catalog number: H3566 )
  11. Laminin 111 (BD biosciences, catalog number: 354232 )
    Note: Currently, it is “Corning, catalog number: 354232”.
  12. Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10 ml (BD biosciences, catalog number: 354230 )
    Note: Currently, it is “Corning, catalog number: 354230”.
  13. Recombinant Mouse Oncostatin M (OSM) Protein (R&D systems, catalog number: 495-MO )
  14. EGF Recombinant Human Protein (Life technologies, catalog number: PHG0311 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: PHG0311”.
  15. Recombinant Human HGF (NS0-expressed) Protein (R&D systems, catalog number: 294-HGN )
  16. Insulin from bovine pancreas (Sigma-Aldrich, catalog number: I5500 )
  17. Insulin/Transferrin/Selenium (ITS) (Life Technologies, catalog number: 41400-045 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 41400-045”.
  18. Dexamethasone (Dex) (Sigma-Aldrich, catalog number: D-4902 )
  19. Anti-CD16/32 antibody (Biolegend, catalog number: 101-301 )
  20. FITC anti-Dlk1 antibody (MBL International, catalog number: D187-4 )
  21. FITC anti-mouse CD326 (Ep-CAM) Antibody (Biolegend, catalog number: 118207 )
  22. APC-Cy7 anti-CD45 (Biolegend, catalog number: 103-115 )
  23. APC-Cy7-TER119 (Biolegend, catalog number: 116-223 )
  24. PE-Cy7-anti-CD31 (Biolegend, catalog number: 102417 )
  25. Paraformaldehyde (PFA)
  26. Hoechst 33258 (Dojindo Molecular Technologies, catalog number: 343-07961 )
  27. Anti-mouse albumin (ALB) antibody (Bethyl laboratories, catalog number: A90-134P )
  28. Anti-cytokeratin 19 (CK19) (Tanimizu et al., 2003)
  29. AlexaFluor 488 conjugated donkey anti-rabbit IgG (Life technologies, catalog number: A-21206 ) and AlexaFluor 555 conjugated donkey anti-goat IgG (Life Technologies, catalog number: A-21432)
    Note: Currently, it is “Thermo Fisher Scientific, Novex™, catalog number: A-21206 and A-21432 ”.
  30. Prolong Gold (Life Technologies, catalog number: P36930 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: P36930”.
  31. L-15 medium (Sigma-Aldrich, catalog number: L4386 )
  32. DMEM/nutrient mixture Ham F-21 (DMEM/F12) medium
  33. Decomplemented Hyclone fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH30910.03 )
    Note: Currently, it is “GE Healthcare, catalog number: SH30910.03”.
  34. Niflumic acid (Sigma-Aldrich, catalog number: N0630 )
  35. Blockace (DS Pharma Biomedical Co, catalog number: UK-B40 )
  36. Cloning ring (ASAHI GLASS CO, catalog number: 11-016-006 )
  37. Trypsin/EDTA (Sigma-Aldrich, catalog number: T4049 )
  38. RNAiMAX (Life Technologies, catalog number: 13778030 )
    Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 13778030”.
  39. RNeasy Mini Kit (QIAGEN, catalog number: 74106 )
  40. ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
  41. Pre-perfusion solution (see Recipes)
  42. Perfusion solution (see Recipes)
  43. L-15 medium (see Recipes)
  44. Hyaluronidase (see Recipes)
  45. Culture medium (see Recipes)

Equipment

  1. Centrifuge (KUBOTA)
  2. FACSAriaII (BD biosciences)
  3. CO2 incubator (The incubator is used to keep culture at 37 °C and under 5% CO2) (SANYO)
  4. Fluorescence microscope (OLYMPUS, model: IX71 ) and digital camera (OLYMPUS, model: DP72 )
  5. Plate reader Mutiskan JX (Labsystems)
  6. ROCK inhibitor (Wako Pure Chemical Industries, catalog number: 257-00511 )
  7. Round-shaped stirring bar (ASONE Corporation, catalog number: 1-5409-01 )

Procedure

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文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Tanimizu, N. (2015). Clonal Culture of Mouse Liver Progenitor Cells. Bio-protocol 5(20): e1624. DOI: 10.21769/BioProtoc.1624.

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分类

干细胞 > 成体干细胞 > 维持和分化

细胞生物学 > 细胞分离和培养 > 细胞分化

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