Materials and Reagents

1. Mice (NCI) (BALB/c or C57BL/6, 4 weeks to 18+ months)
   
2. Isofluorane (USP inhalation vapour, liquid) ( NDC: 57319-559-06 )
   
3. Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, Gibco^®, catalog number: 11965092 )
   
4. Ketaset III ketamine HCl injection (USP 100 mg/ml) (DEA Schedule II Drug) (NDC: 0856-2013-01 )
   
5. 100 mg/ml AnaSed injection xylazine (Lloyd Laboratories, NADA number: 139-236 )
   
6. 0.9% sodium chloride irrigation (USP) (Baxter, catalog number: 2F7124 )
   
7. 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Gibco^®, catalog number: 14190-144 )
   
8. Collagenase D (Roche Diagnostics, catalog number: 11088882001 )
   
9. DNase I (Roche Diagnostics, catalog number: 10104159001 )
   
10. Hank’s balanced salt solution (HBSS) (Life Technologies, Gibco^®, catalog number: 14025 )
    
11. L-Glutamine (200 mM) (Life Technologies, Gibco^®, catalog number: 25030-081 )
    
12. Hepes (1 M) (Life Technologies, Gibco^®, catalog number: 15630-080 )
    
13. Penicillin streptomycin (Life Technologies, Gibco^®, catalog number: 15140-122 )
    
14. RPMI medium 1640 (Life Technologies, Gibco^®, catalog number: 11875-093 )
    
15. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250-100ML )
    
16. BD Golgi Plug^TM (BD, catalog number: 554722 )
    
17. Normal rat serum (Life Technologies, Invitrogen^TM, catalog number: 10710C )
    
18. FITC Anti-mouse CD8a clone 53-6.7 (0.5 mg/ml) (BD, catalog number: 553031 )
    
19. PerCP-cyanine5.5 Anti-mouse CD4 clone RM4-5 (eBioscience, catalog number: 45-0042-82 )
    
20. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
    
21. Sodium azide (AMRESCO, catalog number: 0639 )
    
22. BD Cytofix/Cytoperm^TM fixation and permeabilization solution (BD, catalog number: 554722)
    
23. BD Perm/Wash^TM buffer (BD, catalog number: 554722)
    
24. APC Anti-mouse IFNγ clone XMG1.2 (eBioscience, catalog number: 17-7311-82 )
    
25. Ketamine solution (see Recipes)
    
26. Digestion buffer (see Recipes)
    
27. RP10 (see Recipes)
    
28. FACS buffer (see Recipes)
    
29. Cell surface staining mixture (see Recipes)
    
30. Intracellular staining mixture (see Recipes)
    

Equipment

1. Dessicator (Narang Medical, catalog number: P37.1517P )
   
2. Precision glide needle (25 G x 5/8) (BD, catalog number: 305122 )
   
3. Gauze sponges (4 x 4 inch) (Pro Advantage^® by NDC, catalog number: P157118 )
   
4. 1 ml syringe (BD, catalog number: 309659 )
   
5. 3 ml syringe (BD, catalog number: 309657 )
   
6. Cell strainer (70 µm nylon) (BD, catalog number: 352350 )
   
7. Tissue culture dishes (60 x 15 mm) (BD Biosciences, Falcon^®, catalog number: 353002 )
   
8. 96 well cell culture cluster (round bottom with Lid) (Corning, Costar^®, catalog number: 3799 )
   
9. 5 ml polystyrene round bottom tube (BD Biosciences, Falcon^®, catalog number: 352054 )
   
10. Absorbent pads (Covidien, catalog number: 949 )
    
11. 10 ml syringe (BD, catalog number: 309604 )
    
12. 12 well cell culture cluster (flat bottom with lid) (Corning, Costar^®, catalog number: 3513 )
    
13. 1 ml graduate transfer pipette (Research Products International, catalog number: 147501-1S )
    
14. 15 ml screw cap tube conical (Sarstedt AG, catalog number: 62.554.002 )
    
15. 50 ml screw cap tube conical (Sarstedt AG, catalog number: 62.547.004 )
    
16. Biosafety hood
    
17. Spray bottle with 70% ethanol
    
18. Surgical scissors
    
19. Polystyrene foam
    
20. Pipetman p10
    
21. Pipetman p200
    
22. Pipetman p1000
    
23. CO₂ incubator
    
24. Pipet aid
    
25. Small metal weighing spatula
    
26. Tweezers
    
27. Vortexer
    
28. Shaker rotisserie
    
29. Refrigerated tabletop centrifuge
    
30. Hemocytometer
    
31. Flow cytometer
    

Procedure