Materials and Reagents

1. CO₂ independent medium (Invitrogen, catalog number: 18045054 )
   
2. Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, catalog number: I3390 )
   
3. 10% fetal bovine serum (FBS) (Hyclone/PAA, catalog number: sv143-03 )
   
4. Penicillin-streptomycin (100 Units/ml, 100 μg/ml) (Sigma-Aldrich, catalog number: P11-010 )
   
5. Glutamine (Sigma-Aldrich, catalog number: G75013 )
   
6. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
   
7. Granulocyte-macrophage colony stimulating factor (GM-CSF) (Peprotech, catalog number: 315-03 )
   
8. 40,000 MW Dextran fluorescein (10 mg/ml) (Molecular Probes)
   
9. 40,000 MW Dextran Alexa 647 (10 mg/ml) (Molecular Probes)
   
10. 5 mM EDTA (Invitrogen, catalog number: 15575-038 )
    
11. 1% Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
    
12. Triton X-100 (Sigma-Aldrich, X-100) kept at room temperature
    
13. 1x PBS (see Recipes)
    
14. Conditioned complete medium (see Recipes)
    

Equipment

1. Water bath (37 °C)
   
2. Incubator (37 °C)
   
3. The FACSCalibur flow cytometer (Becton Dickinson)
   
4. Hemocytometer
   
5. Centrifuge
   

Procedure