Immunofluorescence Assay for S Phase Entry Using BrdU Incorporation   

Abstract

Materials and Reagents

1. BrdU (Sigma-Aldrich, catalog number: B5002 )
   
2. Phosphate buffered saline (PBS)
   
3. Methanol (Thermo Fisher Scientific, catalog number: BP1105-4 )
   
4. BSA (Sigma-Aldrich, catalog number: A3803 )
   
5. HCl
   
6. NaN₃
   
7. Triton X-100
   
8. Monoclonal anti-BrdU (Sigma-Aldrich, catalog number: B2531 )
   
9. Alexa 488-conjugated goat anti-mouse immunoglobulin G antibody (Life Technologies, Molecular Probes^®/Alexa Fluor^® 488, catalog number: A-11008 or A-11034 )
   
10. DAPI (make 1 mg ml^-1 stock, 1,000x) (Boeringer Manheim 236 276 )
    
11. Coverglasses (12 mm) (Thermo Fisher Scientific, catalog number: 12-545-82 )
    
12. Fluormount-G^TM Mounting solution (SouthernBiotech, catalog number: 0100-01 )
    
13. PBS-BT solution (see Recipes)
    

Equipment

1. Centrifuges (Beckman Falcon, model: TLS-55 )
   
2. Parafilm
   
3. 6-well plate
   
4. 24-well plate
   

Procedure

1. Split cells onto tissue culture dishes containing coverglasses or chambered slides.
   Notes:
   1. We usually put one coverglass in one well of the 24-well plate; or no more than 2 coverglasses in one well of the 6-well plate, or no more than 5 coverglasses in a 60 mm dish.
      
   2. Pipette up and down or shaking the dish to make sure cells are not concentrating in the center of the dish well. Make sure there are no air bubbles between the coverglasses and the tissue culture dish.
      
2. Grow cells to 70-100% confluency.
   
3. Add 30 μM BrdU to cells. 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.
   
4. Transfer the coverglasses or chambered slides into another tissue plate containing sufficient methanol (-20 °C stock), fix for 10 min or a couple of weeks.
   
5. Carefully transfer the coverglasses or chambered slides from the plates and place cell side up onto secured Parafilm. Wash the coverglasses or chambered slides with 100 µl PBS immediately after transfer, never dry the cells.
   
6. Block with 100 µl PBS-BT for 30 min.
   
7. Incubate 100 µl 2 M HCl, 30 min.
   
8. Wash 2x with 100 µl PBS-BT, each wash for 5 min
   
9. Incubate with 100 µl mouse anti-BrdU (1:100 dilution in PBS-BT), 30 min.
   
10. Wash 3x with 100 µl PBS-BT, each wash for 5 min.
    
11. Incubate with 40 µl second antibody (1:200 dilution in PBS-BT), 30 min.
    
12. Wash 3x with 100 µl PBS-BT, each wash for 5 min.
    
13. Cells were incubated in 40 µl DAPI (1 µg/ml final concentration, 1:1,000 dilute in PBS) for 2 min, and then wash with PBS once.
    
14. Add 5-10 µl mounting solution to a clean microscope slide for each coverglass, place stained coverglass cell side down onto mounting solution from one edge; allow mounting solution to cover the entire surface of the coverglass, avoiding air bubbles.
    
15. Let the mounting solution dry and self-seal for 30 min at RT.
    

Recipes

1. BrdU stock in PBS (pH 7.4) or ddH₂O, filter sterile (MW = 307.1, 20 mM = 0.006 g/ml)
   
2. PBS-BT solution
   10 ml 10x PBS
   3 g BSA (to 3%)
   1 ml 10 % Triton X-100 (to 0.1%)
   1 ml 5% NaN₃
   ddH₂O to 100 ml, store at 4 °C.
   

Acknowledgments

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

References

1. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.