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In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer

结肠直肠癌的体外和体内限制稀释测定法

LA Lauren Agro
CO Catherine A O’Brien

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1659 浏览次数: 25142

评审: HongLok LungJustine MarsolierAnonymous reviewer(s)

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Abstract

The in vitro limiting dilution assay is used to determine the colorectal cancer initiating cell (CC-IC) frequency of a CC-IC enriched suspension culture, grown in growth factor enriched serum free media. The in vivo limiting dilution assay is used to determine the colorectal cancer initiating cell frequency of a primary colorectal cancer sample or an established suspension cell line using immunocompromised murine xenograft models. In vitro and in vivo limiting dilution assays (LDAs) can be used to determine the effect of a specific treatment or genetic knockdown strategy on the initiating cell frequency of a population of CC-ICs or colorectal cancer sample, respectively.

Part I. In vitro CRC LDA

Materials and Reagents

  1. Centrifuge tubes (BD Falcon, catalog numbers: 352096 and 352070 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog numbers: 352096 and 352070”.
  2. 7x sterile, non-tissue culture 96 well U-bottom plates (Corning Inc., FalconTM, catalog number: 351177 )
  3. FACS tubes with 0.35 μm filter (BD Falcon, catalog number: 352235 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog number: 352235”.
  4. Wash media (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320-033 )
  5. 0.4% Trypan blue solution (Life Technologies, catalog number: 15250-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  6. Permanent marker (VWR International, catalog number: 52877-310 )
  7. 0.25% Trypsin (Life Technologies, catalog number: 25200-056 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  8. Penicillin-streptomycin-amphotericin B (1%) (Life Technologies, catalog number: 15240-062 )
    Note: Currently, it is “Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)".
  9. L-glutamine (2 mM) (Invitrogen, catalog number: 25030081 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25030081”.
  10. Non-essential amino acids (1x) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023801 )
  11. Sodium pyruvate (1 mM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH3023901 )
  12. HEPES (1x) (Invitrogen, catalog number: 15630080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630080”.
  13. Heparin sodium salt (4 μg/ml) from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3149-100KU )
  14. Lipids (Sigma-Aldrich, catalog number: L0288 )
  15. Human Epidermal Growth Factor Recombinant (EGF) (20 ng/ml) (Biomartcanada, catalog number: RKP01133 )
  16. Human basic Fibroblast Growth Factor Recombinant (bFGF) (10 ng/ml) (Biomartcanada, catalog number: RKP09038 )
  17. N2 supplement-A (STEMCELL Technologies, catalog number: 07152 )
  18. NeuroCult SM1 Neuronal (STEMCELL Technologies, catalog number: 05711 )
  19. Sytox blue (Life Technologies, catalog number: S11348 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S11348”.
  20. Phosphate-buffered saline (PBS) (Life Technologies, catalog number: 10010023 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10010023”.
  21. 2% fetal bovine serum (FBS) (Life Technologies, catalog number: 26140-111 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 26140-111 ”.
  22. Serum free cell culture media (see Recipes)

Equipment

  1. 37 °C Incubator
  2. Light microscope (Leica Microsystems, model: DM IL LED )
  3. Hemocytometer (Hausser Scientific, catalog number: 1483 )
  4. Pipette aid (INTEGRA Biosciences AG, catalog number: 155 000 )
  5. Pipettes (BD Falcon, catalog number: 357530 )
    Note: Currently, it is “Corning Inc., FalconTM, catalog number: 357530”.
  6. Flow cytometry assisted cell sorter (FACS)

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

Agro, L. and O’Brien, C. A. (2015). In vitro and in vivo Limiting Dilution Assay for Colorectal Cancer. Bio-protocol 5(22): e1659. DOI: 10.21769/BioProtoc.1659.

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分类

癌症生物学 > 癌症干细胞 > 肿瘤形成 > 自我更新

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