Bone Marrow Derived Eosinophil Cultures

Materials and Reagents

1. Mice
   
2. Histopaque 1083 (Sigma-Aldrich, catalog number: 10831 -100ml)
   
3. IMDM with Glutamax-I (Life Technologies, Invitrogen^TM, catalog number: 31980-097 )
   
4. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
   
5. Penicillin/Streptomycin (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
   
6. Stem Cell Factor (Pepro tech, catalog number: 250-03 )
   
7. FLT-3 Ligand (Pepro tech, catalog number: 250-31L )
   
8. Recombinant mouse IL-5 (Pepro tech, catalog number: 215-15 )
   
9. PBS (Life Technologies, Invitrogen^TM, catalog number: 14200-166 )
   
10. Diff-Quick Stain Kit (Thermo Fisher Scientific, catalog numbers: 23-122-929 , 23-122-952 , and 23-122-937 )
    
11. RBC lysis buffer (Sigma-Aldrich, catalog number: R7757 -100ml)
    
12. CCR3 (R&D systems, Catalog number: FAB729F )
    
13. Siglec-F (BD, Pharmingen^TM, Catalog number: 552126 )
    
14. IMDM cell culture media (see Recipes)
    

Equipment

1. 6-well tissue culture plate
   
2. Dissection tools: scalpel, scissors
   
3. 1 ml syringe
   
4. 15 ml sterile centrifuge tube
   
5. Centrifuge
   
6. 37 °C, 5% CO₂ cell culture incubator
   
7. Microscope
   
8. Hemocytometer
   

Procedure