Materials and Reagents

1. Human neuroblastoma cell lines (NGP, NB-1691, IMR-32)
   Note: A cell line is transduced with two different fluorescent proteins, for example GFP (Clontech, catalog number: 632370 ) and tdTomato (Clontech, catalog number: 632534 ), such that there is a GFP positive line and a tdTomato line of the same cell type. In this manner, subpopulations of the same cell type (i.e. GCSF-R positive and GCSF-R negative cells) can be traced. If in vivo monitoring of tumor growth via bioluminescent imaging is desired, cell lines should also be transfected or virally transduced with commercially available vectors, e.g. pGL2-Control Vector (Promega Corporation) to express a luminescent reporter gene. For a detailed description and protocol of in vivo bioluminescent imaging, please refer to Reference 2.
   
2. 4-6 week old female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Taconic, model number: NODSCF , http://www.taconic.com/NODSC)
   
3. 293T cells
   
4. RPMI medium 1640 (Life Technologies, catalog number: 11875-101 )
   
5. 10% (v/v) fetal bovine serum (FBS) (Life Technologies, catalog number: 16000044 )
   
6. 1% (v/v) penicillin/streptomycin (Life Technologies, catalog number: 15140122 ) (100x P/S; final concentration 100 units/ml penicillin and 100 mg/ml streptomycin)
   
7. 1% (v/v) of 100x L-glutamine (Life Technologies, catalog number: 25030081 ) (final concentration 2 mM)
   
8. Phosphate-buffered saline (PBS) (sterile) (Life Technologies, catalog number: 70011069 )
   
9. 0.25% trypsin/EDTA (Life Technologies, catalog number: 25200056 )
   
10. Collagenase I (Sigma-Aldrich, catalog number: C0130 ) [prepare a solution containing 10,000 Collagenase Digestion Unites (CDU/ml) in PBS]
    
11. Dispase II (Roche Diagnostics, catalog number: 04942078001 ) (prepare a solution containing 32 mg/ml Dispase II in PBS)
    
12. DNase I (EMD Millipore, Calbiochem^®, catalog number: 260913 ) (prepare a solution containing 5 MU/ml DNase I)
    
13. FuGENE 6 (Promega Corporation, catalog number: E2691 )
    
14. Opti-MEM Reduced Serum Medium (Life Technologies, catalog number: 31985-062 )
    
15. PE conjugated anti-CD 114 (GCSFR) antibody (BD Biosciences, catalog number 554538 )
    
16. Cell culture medium (see Recipes)
    
17. Sterile FACS buffer (see Recipes)
    
18. PEB Buffer (see Recipes)
    

Equipment

1. Fluorescence-activated cell sorter (e.g. DAKO Cytomation MoFlo 9-color cell sorter)
   
2. 37 °C, 5% CO₂ tissue culture incubator
   
3. Refrigerated centrifuge
   
4. Class 2 biological safety cabinet with laminar flow hood
   
5. 70 μm cell strainer (Thermo Fisher Scientific, catalog number: 22-363-548 )
   
6. T-75 culture flask or 10 cm dish
   
7. Anesthesia machine/chamber with nose cone appropriate for mice (Surgivet or VetEquip)
   
8. Fluorescent microscope
   
9. Surgical instruments
   1. 5.5-in Mayo-Hegar or similar surgical needle holder (Millennium Surgical or Roboz Surgical Instrument)
      
   2. Sterile gloves (Thermo Fisher Scientific)
      
   3. Disposable sterile scalpel blade (#10) (Millennium Surgical or Roboz Surgical Instrument)
      
   4. 27-G needle  
      
   5. Sterile 1-cc slip tip syringe
      
   6. Polysorb 4-0 sutures with RB-1 tapered needle (U.S. Surgical)
      
   7. 9-mm wound clips  (VWR International)
      
   8. Rodent ear tags (National Band & Tag Company)
      

Procedure