Limiting Dilution Assays to Determine Frequencies of Lymphohematopoietic Progenitors

Materials and Reagents

1. Stromal cells (MS5 cells or OP9 cells)
   
2. Growth factors
   a. rm SCF (10 ng/ml) (e.g. R&D systems, catalog number: 455-MC-010 )
   b. rm Flt3-ligand (20 ng/ml) (e.g. R&D systems, catalog number: 427-FL-025 )
   c. rm IL-7 (1 ng/ml) (e.g. R&D systems, catalog number: 407-ML-005 )
   
3. Antibodies
   a. Anti-mouse Mac1 (BD Biosciences, catalog number: 557396 )
   b. Gr1 (BD Biosciences, catalog number: 553128 )
   c. CD19 (BD Biosciences, catalog number: 557399 )
   d. CD45R/B200 (BD Biosciences, catalog number: 553092 )
   e. CD45 (BD Biosciences, catalog number: 550994 )
   
4. Trypsin/EDTA solution (Nacalai Tesque, catalog number: 35554-64 )
   
5. EDTA solution (Nacalai Tesque, catalog number: 14367-74 )
   
6. Alpha Modification of Eagle’s Medium (Corning, cellgro^®, catalog number: 50-012 )
   
7. Minimum Essential Medium Alpha Medium (Gibco®, catalog number: 41061-029 )
   
8. FCS (MP Biomedicals, catalog number: 2916754 )
   
9. L-glutamine (Corning, cellgro^®, catalog number: 61-030 )
   
10. Penicillin-Streptomycin (Nacalai Tesque, catalog number: 26253-84 )
    
11. MS-5 growth media (see Recipes)
    
12. OP-9 growth media (see Recipes)
    

Equipment

1. Cell sorting machines (BD Biosciences, FACSAria^TM) with the automated cell deposition system
   
2. 96-well flat bottom plates
   
3. 10 cm plastic dishes
   
4. CO₂ incubators
   
5. Flow cytometry system
   

Procedure