Materials and Reagents

1. Peripheral blood mononuclear cells (PBMC) [isolated from a buffy coat from a healthy donor using Ficoll-Paque (own pharmacy) density gradient (1.077 g/cm³)]
   
2. Multipotent stromal cells (MSC) from healthy donors
   
3. Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
   
4. Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
   
5. L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
   
6. Fetal calf serum (FCS) (Greiner Bio-One GmbH, catalog number: 758072 )
   
7. Phosphate buffered saline (PBS) (produced by in-house pharmacy)
   
8. M-CSF (Pepro Tech, catalog number: 300-25 )
   
9. CD14 MicroBeads (human) (Miltenyi Biotec, catalog number 130-050-201 )
   
10. Antibodies
    
    1. Anti-CD14 PE (BD Biosciences)
       
    2. Anti-CD206 APC (BD Biosciences)
       
    3. Anti-CD163 PerCP-Cy5.5 (BioLegend)
       
    4. Anti-CD80 PE-Cy7 (BioLegend)
11. Trypsin/EDTA (0.05%, phenol red) (Life Technologies, Invitrogen^TM, catalog number: 25300-054 )
    
12. Culture medium (see Recipes)
    
13. Proliferation medium (see Recipes)
    

Equipment

1. T75 culture flasks
   
2. 6-well plates (Sigma-Aldrich, catalog number: CLS3506 )
   
3. 37 °C, 5% CO₂ cell culture incubator
   
4. Microscope
   
5. Centrifuge
   
6. Hemocytometer (counting chamber)
   
7. MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
   
8. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
   
9. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
   
10. 24-well plates (Sigma-Aldrich, catalog number: CLS3526 )
    

Procedure