Materials and Reagents

1. Isoflurane (Vedco, catalog number: 50201 )
   
2. Hanks' balanced salt solution (HBSS) (Cellgro, catalog number: 20-021-CV )
   Note: 10x solution, diluted to 1x in house in sterile distilled water.
   
3. RPMI-1640 medium (Sigma-Aldrich, catalog number: R8758 )
   
4. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11055H )
   
5. Penicillin/streptomycin (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
6. Collagenase type IV (Worthington Biochemical, catalog number: 4188 )
   
7. DNase I (Sigma-Aldrich, catalog number: DN25 )
   
8. Percoll (GE Healthcare, catalog number: 17-0891-01 )
   
9. Phosphate buffered saline (PBS)
   
10. Anti-CD45-allophycocyanin (APC) and anti-CD11b-phycoerythrin (PE) antibodies (both from BD, catalog numbers: 559864 and 557397 , respectively)
    
11. Anti-CD8-APC (clone Ly-; 2) (BD, catalog number: 553035 )
    
12. Anti-CD4-PE (clone L3T4) (BD, catalog number: 553049 ) antibodies
    
13. Complete RPMI-1640 medium (see Recipes)
    
14. Collagenase and DNase I solution (see Recipes)
    
15. 100% Percoll (see Recipes)
    
16. 70% Percoll (see Recipes)
    
17. 30% Percoll (see Recipes)
    

Equipment

1. Scissors and forceps
   
2. 10 ml and 50 ml disposable plastic syringe (BD)
   
3. 18 G needle (BD)
   
4. Steel mesh (250 m) (VWR International, catalog number: AA13478-RR )
   
5. 37 °C water bath
   
6. Table top refrigerated centrifuge
   
7. High-speed centrifuge (Beckman Coulter, model: J2-21 )
   
8. 50 ml screw-cap tube
   
9. LSRII Flow cytometer (BD)
   
10. Low-speed table top centrifuge (1,000-3,000 rpm)
    

Procedure