Natural Killer Cell Transfer Assay

Materials and Reagents

1. Lympholyte-M (Cedarlane, catalog number: CL5035 )
2. Source of mouse NK cells: C57Bl/6 mice (Charles Rivers, strain code: 027 )
   
3. NK-deficient mice (Jackson Labs, strain name: B6.129S4-Il2rg^tm1Wjl/J, strain number: 003174 )
   
4. Gluteraldehyde
   
5. Potassium ferrocyanide
   
6. Potassium ferricyanide
   
7. Bovine serum albumin (BSA)
   
8. 100x non-essential amino acids
   
9. Fetal bovine serum (FBS)
   
10. Sodium pyruvate
    
11. Deoxycholate
    
12. B16F10lacZ melanoma tumor cells, grown in complete DMEM (Dr. K. Graham, London Regional Health Centre, London ON)
    
13. 1x sterile PBS (Hyclone, catalog number: 21-031-CV )
    
14. X gal (Bioshop Canada Inc., catalog number: XGA001-1 )
    
15. Running buffer (Miltenyi Biotech, catalog number: 130-091-221 )
    
16. Washing buffer (Miltenyi Biotech, catalog number: 130-092-987 )
    
17. AutoMACS pro columns (Miltenyi Biotech, catalog number: 130-021-101 )
    
18. RPMI 1640 (Hyclone, catalog number: SH300027.01 )
    
19. AutoMACS buffer (see Recipes)
    
20. Complete RPMI (see Recipes)
    
21. NK cell media (see Recipes)
    
22. LacZ staining solutions (see Recipes)
    

Equipment

1. 50 ml tubes (BD Biosciences, Falcon^®, catalog number: 352098 )
   
2. 15 ml tubes (BD Biosciences, Falcon^®, catalog number: 352096 )
   
3. Insulin syringe 27G1/2” (Terumo Medical Corporation, catalog number: SS05M2713 )
   
4. AutomacsPro Separator (Miltenyi biotech, model: 130-092-545 )
   
5. Incubator (5% CO₂, 37 °C) (Sanyo)
   
6. Centrifuge (when parameters of brakes are unspecified, maximal acceleration and deceleration are used), (Thermo Fisher Scientific, model: ST40R )
   
7. Dissection instruments (small forceps, scissors)
   
8. Light microscope (Leica)
9. Cell strainers 70 µm (Thermo Fisher Scientific, catalog number: 22363548 )
   
10. DX5 (CD49b) microbeads (Miltenyi Biotech, catalog number: 130-052-501 )
    

Procedure