Materials and Reagents

1. Mice (NCI) (Balb/c or C57BL/6, 4 weeks to 18+ months)
   
2. Vero E6 cells (ATCC, catalog number: CRL-1586 )
   
3. Isofluorane (USP inhalation vapour; liquid) ( NDC: 57319-559-06 )
   
4. Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, Gibco^®, catalog number: 11965092 )
   
5. 100 mg/ml ketaset III ketamine HCl injection (USP) (DEA Schedule II Drug) (NDC: 0856-2013-01 )
   
6. 100 mg/ml AnaSed injection xylazine (Lloyd Laboratories, NADA number: 139-236 )
   
7. 0.9% sodium chloride irrigation (USP) (Baxter, catalog number: 2F7124 )
   
8. 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Gibco^®, catalog number: 14190-144 )
   
9. Formaldehyde solution (Sigma-Aldrich, catalog number: 252549 ) (37 wt.% in H₂O)
   
10. Crystal violet (Sigma-Aldrich, catalog number: C0775 )
    
11. 25 mM DMEM with hepes (Life Technologies, Gibco^®, catalog number: 32430 )
    
12. 200 mM L-Glutamine (Life Technologies, Gibco^®, catalog number: 25030-081 )
    
13. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
    
14. MEM non-essential amino acids (Life Technologies, Gibco^®, catalog number: 11140-050 )
    
15. 50 mg/ml gentamicin sulfate (Lonza, catalog number: 17-518Z )
    
16. Penicillin streptomycin (Life Technologies, Gibco^®, catalog number: 15140-122 )
    
17. DMEM (Life Technologies, Gibco^®, catalog number: 12100-046 )
    
18. Agarose (optimized grade) (Research Products International, catalog number: A20090 )
    
19. Ketamine solution (see Recipes)
    
20. Culture media (see Recipes)
    
21. Overlay media (see Recipes)
    

Equipment

1. Biosafety hood in a biosafety level 3 facility
   
2. Absorbent pads (Covidien, catalog number: 949)
   
3. 2 ml micro tube (Sarstedt AG, catalog number: 72.694.006 )
   
4. 5 ml syringe (BD, catalog number: 309646 )
   
5. 75 cm² cell culture flask (canted neck, 0.2 µM vent cap) (Corning, catalog number: 430641 )
   
6. 12 well cell culture cluster (flat bottom with lid) (Corning, Costar^®, catalog number: 3513 )
   
7. 48 well cell culture cluster (flat bottom with lid) (Corning, Costar^®, catalog number: 3548 )
   
8. 10 ml stripette (Corning, Costar^®, catalog number: 3548)
   
9. 35 ml closed tissue grinder system (Fisher Scientific, catalog number: 02-542-08 )
   
10. Dessicator (Narang Medical, catalog number: P37.1517P )
    
11. Gauze Sponges (4 x 4 inch) (Pro Advantage^® by NDC, catalog number: P157118 )
    
12. 1 ml syringe (BD, catalog number: 309659 )
    
13. Precision glide needle (25 G x 5/8) (BD, catalog number: 305122 )
    
14. Spray bottle with 70% ethanol
    
15. Surgical scissors
    
16. Polystyrene foam
    
17. Pipetman P200 micropipette
    
18. Pipetman P1000 micropipette
    
19. CO₂ incubator
    
20. Pipet aid
    
21. Small metal weighing spatula
    
22. Tweezers
    
23. -80 °C freezer
    

Procedure