Materials and Reagents

1. Mice: Donor 8-12 week-old mice and sex-matched receptors. We always used C57BL/6 mice but it should work for any mice strain.
   
2. Red blood lysing buffer Hybri-Max (Sigma-Aldrich, catalog number: R7757 )
   
3. PBS (Life Technologies, Gibco^®, catalog number: 10010-023 )
   
4. EDTA (Life Technologies, Gibco^®, catalog number: 15575-020 )
   
5. Fetal Bovine Serum (FBS) (PAA Laboratories GmbH, catalog number: A15-201 )
   
6. Anti-Ly-6G Microbead kit (Miltenyi Biotec, catalog number: 130-092-332 )
   
7. 0.4% Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
   
8. PE-Cy7 labeled anti mouse Ly-6G (eBioscience, catalog number: 25-5931 )
   
9. FITC labeled anti mouse CD11b (eBioscience, catalog number: 11-0112 )
   
10. Isoflurane
    
11. Flushing/washing buffer (see Recipes)
    
12. MACS buffer (modified) (see Recipes)
    

Equipment

1. LS column (Miltenyi Biotec, catalog number: 130-042-401 )
   
2. Laminar Flow Cabinet
   
3. MACS separators
   
4. Centrifuge Eppendorf 5810R (Eppendorf, model: 5811000.010 )
   
5. Centrifuge rotor for plates A-4-62 (Eppendorf, model: 5810709.008 )
   
6. Optic microscope
   
7. Counting Neubauer chamber
   
8. Flow cytometer BD FACS Canto II
   
9. Automatic pipettes (full range volumes)
   
10. Sterile forceps and scissors
    
11. Tips (full range volumes)
    
12. Microtubes (1.5 ml)
    
13. Tubes (15 and 50 ml)
    
14. Culture dishes (45 mm)
    
15. Conventional Insulin 1 ml syringes with detachable needle (25G) (BD Biosciences, catalog number: 329651 )
    
16. Low-dead volume insulin 1 ml syringes (29G) (BD Biosciences, catalog number: 329410 )
    
17. Cell strainer 40 μm (BD Biosciences, catalog number: 352340 )
    
18. Bell jar
    
19. 0.2 μm filter
    

Procedure