Protocol to Determine Mitotic Index by FACS    

Abstract

Materials and Reagents

1. Phosphate buffered saline (PBS)
   
2. Ethanol
   
3. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
   
4. BSA (Sigma-Aldrich, catalog number: A3803 )
   
5. MPM-2 monoclonal antibody
   
6. Anti-phospho-Histone H3 (Thr11) (EMD Millipore, catalog number: 06-570 )
   
7. Anti-phospho-Ser/Thr-Pro, MPM-2 (EMD Millipore, catalog number: 05-368 )
   
8. Alexa 488-conjugated goat anti-mouse immunoglobulin G antibody (Life Technologies, Molecular Probes^®/Alexa Fluor^® 488, catalog number: A-11008 or A-11034 )
   
9. RNase A (Sigma-Aldrich, catalog number: R4642 )
   
10. PI (Sigma-Aldrich, catalog number: P-4170 )
    

Equipment

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

Procedure

Note: All spins are done at 2,000 rpm for 5 min.

1. After collecting the cells (of your choice), wash them with 500 μl of PBS once, resuspend in 150 μl of PBS and then add 350 μl ethanol. Mix and store cells at 4 °C for at least 1 h.
   
2. Spin and remove ethanol. Resuspend cells in 500 μl of PBS containing 0.25% Triton X-100 and incubate on ice for 15 min.
   
3. After centrifugeation, the cell pellet was suspended in 100 μl of PBS containing 1% BSA and 0.25 μg of Histone H3 monoclonal antibody or 0.06 µg MPM-2 monoclonal antibody and incubated for 1 h at room temperature (RT).
   Note: After this step, cell pellets become loose even after centrifugation, therefore it is better to use a pipet to remove the solutions rather than using an aspirator.
   
4. Spin and wash with 150 μl of PBS containing 1% BSA once.
   
5. Resuspend cells in Alexa 488-conjugated goat anti-mouse immuneoglobulin G antibody diluted at a ratio of 1:300 in 100 μl of PBS containing 1% BSA and incubate at RT in the dark for 30 min.
   
6. After centrifugation, resuspend cells in 500 μl of PBS containing 10 μg ml^-1 RNase A and 20 μg/ml PI, transfer to FACS tubes and incubate at RT in the dark for 30 min.
   
7. Take sample to FACS immediately or store at 4 °C until FACS analysis.
   

Acknowledgments

This protocol was developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

1. Field, C. M., Wuhr, M., Anderson, G. A., Kueh, H. Y., Strickland, D. and Mitchison, T. J. (2011). Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. J Cell Sci 124(Pt 12): 2086-2095.
   
2. Preuss, U., Landsberg, G. and Scheidtmann, K. H. (2003). Novel mitosis-specific phosphorylation of histone H3 at Thr11 mediated by Dlk/ZIP kinase. Nucleic Acids Res 31(3): 878-885.
   
3. Qian, J., Lesage, B., Beullens, M., Van Eynde, A. and Bollen, M. (2011). PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting. Curr Biol 21(9): 766-773.