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, Invitrogen^TM, 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