Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)   

Abstract

Materials and Reagents

1. BrdU (Sigma-Aldrich, catalog number: B5002 )
   
2. RNase A (Sigma-Aldrich, catalog number: R4642 )
   
3. Tween 20 (Thermo Fisher Scientific, catalog number: BP337-500 )
   
4. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
   
5. BSA (Sigma-Aldrich, catalog number: A3803 )
   
6. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
   
7. Monoclonal anti-BrdU (Sigma-Aldrich, catalog number: B2531 )
   
8. Goat-anti-Mouse IgG (Whole molecule) FITC conjugate (Sigma-Aldrich, catalog number: F0257 )
   
9. Sodium Tetraborate (Na₂B₄O₇•10H₂O) (Sigma-Aldrich, catalog number: B9876 )
   
10. 0.05% Trypsin-EDTA (Life Technologies, Gibco^®, catalog number: 25300-062 )
    
11. Phosphate buffered saline (PBS)
    
12. EtOH
    
13. HCl
    
14. Sodium tetraborate (see Recipes)
    
15. PBS/ 1% BSA (see Recipes)
    
16. PI stock solution (see Recipes)
    

Equipment

1. Centrifuges (Beckman Falcon, TLS-55 )
   
2. Fluorescence Activated Cell Sorting (FACS) machine
   
3. FACS tubes (BD Biosciences, Falcon^®, catalog number: 352054 )
   

Procedure

1. Culture cells under optimum growing conditions (log growth phase at critical cell density). Add BrdU at a final concentration of 30 μM. Incubate for 30-60 min.
   Note: BrdU is light sensitive and should be added in the dark. Cells pulsed with BrdU may be photosensitive--incubations should be in the dark as well. Time of pulse and BrdU concentration variable with cell type and doubling time. Ranges from 15 min to 2 h, and from 10 μM to 100 μM. For example: We treat mouse embryonic fibroblast cells at 30 µM for 60 min.
   
2. Remove BrdU media. Rinse once with PBS.
   
3. Trypsinize and harvest cells.
   
4. Permeabilize cells: Rsuspend pellet in 0.3 ml PBS, agitate gently, then add 0.7 ml ice-cold 100% EtOH slowly. Mix gently with a 1 ml glass transfer pipette. Store at 4 °C at least 1 h.
   Note: The cell concentration following permeabilization should be approximately 10⁶ cells/sample. Samples can be stored in EtOH for up to 2 weeks at 4 °C.
   
5. Pellet cells. Aspirate supernatant completely.
   Note: All pellet cells below at 4,000 rpm, 2 min.
   
6. Add 0.5 ml 2 N HCl/0.5% Triton X-100 and incubate 30 min at room temperature (RT).
   
7. Pellet cells and remove supernatant. Resuspend cells in 0.5 ml 0.1 M sodium tetraborate for 2 min. Pellet cells.
   
8. Wash cells once with 150 µl PBS/ 1% BSA.
   
9. Resuspend cells in 50 μl 0.5% Tween 20/1% BSA/PBS. Add 10-20 μl (1 μg/10⁶ cells) α-BrdU (mAb) and incubate for 1 h at RT.
   
10. Pellet cells and wash cells once with 150 µl PBS/ 1% BSA.
    
11. Pellet cells and resuspend in 50 μl 0.5% Tween 20/1% BSA/PBS. Add 5 μl (1 μg/10⁶ cells) Goat anti-Mouse IgG-FITC. Incubate 30 min at RT.
    
12. Pellet cells and transfer to FACS tubes.
    
13. Resuspend pellet in 0.5 ml PBS containing 10 μg/ml RNase A and 20 μg/ml PI stock solution.
    
14. Leave samples at RT for 0.5 h in the dark.
    
15. FACS immediately or store at 4 °C.
    Notes: Flow cytometry setup controls needed:
    1. Cells stained with PI alone.
       
    2. Cells with no BrdU pulse, and only the antibodies added.
       

Recipes

1. 0.1 M sodium tetraborate (pH 8.5)
   2 N HCl
   0.5% Trition X-100 (0.05 ml)
   1.67 ml HCl in 8.28 ml ddH₂O.
   
2. PBS/ 1% BSA
   0.1 mg BSA in 10 ml PBS.
   
3. 0.5% tween 20/1% BSA/PBS
   0.05 ml Tween 20
   0.1 mg BSA in 9.5 ml PBS.
   
4. 1 mg/ml PI stock solution
   10 mg PI
   10 ml ddH₂O
   5. BrdU stock in PBS pH 7.4 or ddH₂O, filter sterile (MW = 307.1, 20 mM = 0.006 g/ml)
   

Acknowledgments

This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.

References

1. Hoy, C. A., Seamer, L. C. and Schimke, R. T. (1989). Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram. Cytometry 10(6): 718-725.