Materials and Reagents

1. Splenocyte suspension
   1. Eight- to twelve-week old Balb/c mice strain
      
   2. RPMI 1640 (Life Technologies, Gibco^®, catalog number: 22400-089 )
      
   3. Phosphate buffer saline (PBS) (see Recipes)
      
   4. Sterile red blood lysis buffer (ACK buffer) (see Recipes)
      
      
2. Cell lines
   1. 4T1 cell line (ATCC)
      4T1 is a highly metastatic stage IV murine breast cancer cell line that lacks estrogen and progesterone nuclear receptors and that spontaneously metastasizes to lung, brain and bone.
      
   2. RPMI 1640
      
   3. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 10438-026 )
      
   4. 100x Antibiotic-antimycotic (Life Technologies, Invitrogen^TM, catalog number: 15240062 )
      
      
3. Determination and purification of CD4⁺ Treg and naïve T cells
   1. Allophycocyanin (APC)-conjugated anti-CD4 antibody (clone GK1.5) (eBioscience, catalog number: 17-0041 )
      
   2. Alexa Fluor 488-conjugated anti-CD25 antibody (clone PC61.5) (eBioscience, catalog number: 53-0251 )
      
   3. Phycoerythrin (PE) -conjugated anti-CD62L antibody (clone MEL-14) (eBioscience, catalog number: 12-0621 )
      
   4. PE-conjugated anti-Foxp3 antibody (clone FJK-16s) (eBioscience, catalog number: 12-5773 )
      
   5. Fix/Perm buffer (eBioscience, catalog number: 00-5123 , 00-5223 )
      
   6. 10x Permeabilization Buffer (eBioscience, catalog number: 00-8333 )
      
   7. Dynal^® Mouse CD4 Cell Negative Isolation Kit (Life Technologies, Invitrogen^TM, catalog number: 114-15D )
      
   8. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco^®, catalog number: 10438-026)
      
   9. FACS buffer (see Recipes)
      
   10. Sorted cells collection medium (see Recipes)
       
       
4. Differentiation of Treg in vitro
   1. NA/LE Hamster anti-mouse CD3ε monoclonal antibody (clone 145-2C11) (BD, catalog number: 553057 )
      
   2. NA/LE Hamster anti-mouse CD28 monoclonal antibody (clone 37.51) (BD, catalog number: 553294 )
      
   3. Antibiotic-antimycotic (Life Technologies, Invitrogen^TM, catalog number: 15240062)
      
   4. RPMI 1640 supplemented with 50 µM β-mercaptoethanol and antibiotic-antimycotic (Life Technologies, Invitrogen^TM, catalog number: 15240062)
      
   5. Recombinant hTGFβ₁ (R&D Systems, catalog number: 100-B ) (see Recipes)
      
   6. Recombinant mIL-2 (R&D Systems, catalog number: 402-ML ) (see Recipes)
      

Equipment

1. One milliliter syringe (BD, catalog number: 309628 )
   
2. Sterile scissors
   
3. P60 petri dishes (Greiner Bio-One GmbH, catalog number: 628160 )
   
4. Sterile 70-μm filter (BD, catalog number: 352350 )
   
5. Syringe filter (0.22 μm) (Corning, catalog number: 431219 )
   
6. FACSAria cell sorter
   
7. FACSAria II (BD, catalog number: 642510 )
   
8. Airstream Class II BSC (ESCO Corporation)
   
9. 15 ml conical tubes (BD, catalog number: 352095 )
   
10. 5 ml polystyrene round bottom tubes (BD, catalog number: 352052 )
    
11. Twenty four well plates (Greiner Bio-One GmbH, catalog number: 662160 )
    
12. Centrifuge 5810R (Eppendorf, catalog number: 5811 000,622 )
    
13. Dynal MCP-L (Life Technologies, Invitrogen^TM, catalog number: 120.21D )
    
14. CO₂ incubator
    

Procedure