Hoechst/BrdUrd/PI method for cell proliferation I always use 100 mM Tris, pH 7.4, 154 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% (vol/vol) Nonidet-P40, 0.2% (weight/vol) BSA, 1.2 µg/ml Hoechst 33258. Prepare weekly from a 10 x concentrated stock solution in deionised water. I then add PI solution at 200 µg/ml in distilled water to give a final concentration of 2µg/ml. The permeabilisation procedure is carried out on ice and the cells are also stored on ice before analysis. Rabinovitch, P.S., Kubbies, M., Chen, Y.C., Schindler, D., and Hoehn, H., (1988). Exp. Cell Res., 74, 309. Ormerod, M.G., and Kubbies, M. (1992). Cytometry, 13, 678. The method is discussed fully in M.G. Ormerod & M. Poot. in 'Flow Cytometry: A Practical Approach'. 3rd Edition. Edited by M.G. Ormerod. IRL Practical Approach series. Oxford University Press, 2000. Michael Ormerod 34 Wray Park Road Reigate RH2 ODE Telephone: +44 (0)1737 241726 FAX: +44 (0)1737 226736 Mobile telephone: 07802 293242 Web site: http://ourworld.compuserve.com/homepages/Michael_Ormerod I see no reason why themethod should not work after fixation in ethanol, but I have never tried this. The standard mwthod os so straight forward I have never seen a good reason for changing it. Message text written by "Lindemann, Carsten" > I have two questions about the protocol for the BrdU/Hoechst Quenching assay. Is it sufficient to permeabilize cells by using NP40 (0,5%) buffer in order to stain nuclear DNA? Is fixation with ethanol recommended or does Eth disturb Quenching of Hoechst dye? Thanks for any comments Carsten Lindemann EUFETS GmbH Germany <
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