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FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles

基于FACS方法的小鼠后肢肌肉中卫星细胞的分离

Anastasia Gromova Anastasia Gromova
Matthew T Tierney Matthew T Tierney
Alessandra Sacco Alessandra Sacco

发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1558 浏览次数: 13445

评审: Jalaj GuptaAnonymous reviewer(s)

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Abstract

Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal muscle repair and muscle stem cell pool repopulation.

Materials and Reagents

  1. Laboratory mice, C57BL/6 strain, 2-4 months of age
    Note: All protocols have been approved by the Sanford-Burnham Medical Research Institute Animal Care and Use Committee.
  2. Isoflurane (Santa Cruz Biotechnology, catalog number: sc-363629Rx )
  3. Ham’s F-10 media (Life Technologies, Gibco®, catalog number: 11550-043 )
  4. Horse serum (Life Technologies, Gibco®, catalog number: 16050-122 )
  5. Collagenase type II (Life Technologies, Gibco®, catalog number: 17101-015 )
  6. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
  7. Phosphate buffered saline (PBS) (pH 7.4) (Life Technologies, catalog number: 10010-023 )
  8. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  9. Goat serum (Life Technologies, Gibco®, catalog number: 16210-072 )
  10. Antibodies
    1. Biotin CD45 (clone 30-F11, 0.5 mg/ml) (BD Biosciences, catalog number: 553078 )
    2. Biotin CD31 (clone 390, 0.5 mg/ml) (eBioscience, catalog number: 13-0311-85 )
    3. Biotin CD11b (clone M1/70, 0.5 mg/ml) (BD Biosciences, catalog number: 553309 )
    4. Biotin Ly-6A/E-Sca1 (clone E13-161.7, 0.5 mg/ml) (BD Biosciences, catalog number: 553334 )
    5. 7-integrin/PE or FITC (clone R2F2, 1 mg/ml) (AbLabs, catalog number: 53-0010-01 )
    6. CD34/Alexa Fluor 647 (clone RAM34, 0.2 mg/ml) (BD Biosciences, catalog number: 560230 )
    7. Streptavidin APC-Cy7 (0.2 mg/ml) (BD Biosciences, catalog number: 554063 )
    8. Streptavidin microbeads (Miltenyi Biotec, catalog number: 130-048-101 )
    9. FxCycle Violet stain (DAPI, 0.5 mg/ml) (Life Technologies, InvitrogenTM, catalog number: F10347 )
  11. Media (see Recipes)
  12. Stock collagenase type II solution (see Recipes)
  13. Digestion media I (see Recipes)
  14. Digestion media II (see Recipes)
  15. FACS buffer (see Recipes)

Equipment

  1. Isoflurane vaporizer, supply gas (oxygen), flowmeter and induction chamber
  2. TC-treated culture dish (10 cm) (Corning Incorporated, catalog number: 430167 )
  3. Polypropylene centrifuge tubes, sterile (50 ml) (BD Biosciences, catalog number: 352098 )
  4. Polypropylene centrifuge tubes, sterile (15 ml) (BD Biosciences, catalog number: 352096 )
  5. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  6. Cell strainers (70 μm) (Thermo Fisher Scientific, catalog number: 22363548 )
  7. Syringes with 20G x 1’’ needles (BD Biosciences, catalog number: 309644 )
  8. 5 ml FACS round-bottom tubes (BD Biosciences, Falcon®, catalog number: 352063 )
  9. Tools for muscle dissection and mincing: Razor blades and/or small scissors, forceps
  10. Tissue culture laminar flow hood
  11. Shaking water bath
  12. Standard temperature-controlled table-top centrifuge
  13. MACS magnetic separator and multi-stand (Miltenyi Biotec, catalog number: 130-042-302 and 130-042-303 )
  14. Flow cytometer [, (FACSAria cell sorter equipped with 488, 405 and 633 nm lasers) (BD Biosciences)
    Note: Color combinations can be adjusted to match the laser combinations available.

Software

  1. FlowJo software (Tree Star, optional)

Procedure

English
中文翻译

文章信息

版权信息

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

如何引用

Gromova, A., Tierney, M. T. and Sacco, A. (2015). FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles. Bio-protocol 5(16): e1558. DOI: 10.21769/BioProtoc.1558.

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分类

干细胞 > 成体干细胞 > 肌肉干细胞

细胞生物学 > 细胞分离和培养 > 细胞分离

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