EML Erythroid and Neutrophil Differentiation Protocols

Materials and Reagents

1. IMDM (powder or liquid)
   
2. Horse serum (HS) (may require batch testing if low cell viability)
   
3. Foetal bovine serum (FBS)
   
4. L-Glutamine
   
5. Penicillin/Streptomycin (P/S)
   
6. SCF-conditioned medium (SCF-CM) (see Notes)
   
7. Recombinant human erythropoietin (e.g. epoietin alpha, Amgen) (obtained through hospital pharmacy under appropriate local guidelines)
   
8. Recombinant mouse interleukin-3 (IL-3) (e.g. Pepro Tech, catalog number: 213-13 )
   
9. Recombinant mouse granulocyte-monocyte colony-stimulating factor (GM-CSF) (e.g. Pepro Tech, catalog number: 315-03 )
   
10. All-Trans Retinoic Acid (ATRA) (e.g. Sigma-Aldrich, catalog number: 302-79-4 , reconstitute in ethanol)
    
11. Culture supplements and antibiotics (L-Glutamine and Penicillin/Streptomycin)
    
12. Monoclonal anti-mouse antibodies for flow cytometry
    1. C-kit/CD117 (clone 2B8) (e.g. PE-Cy7, eBioscience, catalog number: 25-1171 ) (suggested use at 1:100 dilution)
       
    2. CD34 (clone RAM34) (e.g. Alexa-Fluor 647, eBioscience, catalog number:  51-0341 ) (suggested use at 1:100 dilution)
       
    3. Mac-1/CD11b (clone M1/70) (e.g. PE, eBioscience, catalog number: 12-0112 ) (suggested use at 1:100 dilution)
       
    4. Gr1/Ly6C (clone RB6-8C5) (e.g. FITC, eBioscience, catalog number: 11-5931 ) (suggested use at 1:100 dilution)
       
    5. Sca-1/Ly6A/E (clone D7) (e.g. Pacific blue, BioLegend, catalog number: 122520 ) (suggested use at 1:50 dilution)
       Note: Stain on ice, in culture medium, for 20 min; wash with 10-20x volume of medium; pellet cells at 400 x g for 5 min. Re-suspend in 300-500 μl of medium for FACS analysis.
13. Trypan blue
    

Equipment

1. T175 tissue culture flasks with filtered cap (for production of conditioned medium)
   
2. T75 or T25 tissue culture flasks with filtered cap, or 6-well plates
   Note: For EML cultures, the size of the tissue culture vial is determined by the cell number seeded at the cell densities indicated in the protocol.
   
3. Cell culture incubator
   

Procedure