Materials and Reagents

1. Anti-Mouse CD45.1-APC (eBiosciences, catalog number: 17-0453-81 )
   
2. Rat anti-mouse CD8-PerCpCy5.5 (Biolegend, catalog number: 100733 )
   
3. Anti-mouse CD3-PE (Biolegend, catalog number: 100307 )
   
4. Anti-mouse CD19-PE (Biolegend, catalog number: 115507 )
   Note: The above antibodies have been tested by the authors but may be substituted with antibodies conjugated with other fluorochromes obtained from other manufacturers.
   
5. B3Z CD8⁺ T cell hybridoma is a kind gift from Dr. PK Srivastava (University of Connecticut Health Center)
   
6. RPMI Medium 1640 + L-Glutamine (Life Technologies, catalog number: 11875-093 )
   
7. Phosphate buffered saline (PBS) (Life Technologies, Gibco^®, catalog number: 10010-023 )
   
8. Fetal bovine serum (FBS) (Hyclone, catalog number: SH303397.03 )
   
9. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 )
   
10. β-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
    
11. Antibiotic-Pen-Strep (Life Technologies, Gibco^®, catalog number: 15140-122 )
    
12. Nonidet-P40 (NP40 (US Biological, catalog number: N3500 )
    
13. CPRG Chlorophenol red-D-galactopyranoside (Roche Diagnostics, catalog number: 10884308001 )
    
14. Endograde ovalbumin (Biovendor, catalog number: 321000 )
    
15. CFSEcarboxyfluorescein diacetate, succinimidyl ester (Life Technologies, Molecular Probes^®, catalog number: C34554 )
    
16. CD8a (Ly-2) microbeads (Miltenyi Biotec, catalog number: 130-049-401 )
    
17. Anti-PE MicroBeads (Miltenyi Biotec, catalog number: 130-048-801 )
    
18. CD11c MicroBeads, mouse (Miltenyi Biotec, catalog number: 130-052-001 )
    
19. Recombinant GM-CSF (Pierce Biotechnology, catalog number: RMGMCSF20 )
    
20. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
    
21. L-glutamine
    
22. BMDC medium (see Recipes)
    
23. Soluble ovalbumin protein solution (see Recipes)
    
24. FACS buffer (see Recipes)
    
25. MACS buffer (see Recipes)
    
26. CPRG solution (see Recipes)
    
27. CPRG lysis solution (see Recipes)
    
28. CFSE dye solution (see Recipes)
    

Equipment

1. LSR II flow cytometer (Becton-Dickinson)
   
2. MACS Multi Stand (Mitenyi Biotec, catalog number: 005126 )
   
3. Microplate reader (Bio-Rad Laboratories)
   
4. FlowJo software (Tree Star)
   
5. 96-well plates round bottom (Corning Incorporated, catalog number: 3799 )
   
6. 96-well plates flat bottom (Cell Star, catalog number: 655180 )
   
7. 100 mm TC-Treated culture dishes (Corning Incorporated, catalog number: 430293 )
   
8. 40 μm cell strainer (BD Biosciences, Falcon^®, catalog number: 352340 )
   
9. FACS tube, polystyrene (BD Biosciences, Falcon^®, catalog number: 352054 )
   Note: Perform the entire method with sterile pyrogen free dishes or plates, pipettes, tips, microfuge and conical tubes. Autoclave and filter all the buffers through 0.22 μm filter.
   
10. Incubator
    
11. 10 mm culture dish
    
12. 5 ml conical tube
    

Procedure