Materials and Reagents

1. RPMI 1640 (Life Technologies, Gibco^®, catalog number: 22400-089 )
   
2. Trypan blue 0.4% solution (Lonza, catalog number: 17-942E )
   
3. Myeloid-Derived Suppressor Cell Isolation Kit (mouse) (Miltenyi Biotec, catalog number: 130-094-538 )
   
4. Pan T Cell Isolation Kit II, mouse (Miltenyi Biotec, catalog number: 130-095-130 )
   
5. Antibodies for flow cytometry: Ly-6G-FITC (Gr1, RB6-8C5), CD11b-PE (M1/70), and CD3-FITC (17A2) (Biolegend, catalog numbers: 108405 , 01207 , and 100203 respectively)
   
6. MACS^® BSA Stock Solution (Miltenyi Biotec, catalog number: 130-091-376 )
   
7. AutoMACS^® Rinsing Solution (Miltenyi Biotec, catalog number: 130-091-222 )
   
8. Phosphate buffer saline (PBS) (see Recipes)
   
9. Sterile red blood cell lysis buffer (RBC lysis buffer) (see Recipes)
   
10. MACS buffer (see Recipes)
    

Equipment

1. LS column (Miltenyi Biotec, catalog number: 130-042-401 )
   
2. MidiMACS Separator (Miltenyi Biotec, catalog number: 130-042-302 )
   
3. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
   
4. Wide field microscope (Nikon Diaphot Phase Contrast Inverted Laboratory Microscope, catalog number: 805426 )
   
5. Sterile forceps and scissors
   
6. Flow cytometer
   
7. 1 ml syringes (29 G) (BD Biosciences, catalog number: 329410 )
   
8. Sterile Cell strainers 70 μm (BD Biosciences, catalog number: 352350 )
   
9. 15 ml conical tubes (BD Biosciences, catalog number: 352095 )
   
10. Tabletop centifuge 
    
11. Cell culture centrifuge
    
12. Sterile culture hood
    
13. Hemocytometer
    
14. 60 mm cell culture dish
    

Procedure