From: Reece, Lisa (lreece@utmb.edu)
Date: Wed Nov 11 1998 - 18:06:23 EST
-----Original Message----- From: Reece, Lisa Sent: Wednesday, November 11, 1998 5:03 PM To: cyto-inbox Subject: RE: HeLa, K562 & cell cycle... Mark, As an aside to Derek's message, you must also be sure that the PI solution you use includes RNase. Remember that PI is a nucleic acid stain and will bind to RNA as well as DNA and this could result in broaden peaks as well. Good luck, Lisa Reece Molecular Cytometry Unit Univ. of Texas Medical Branch Galveston, TX USA Office: (409)747-1932 FAX: (409)772-6527 E-mail: lreece@utmb.edu -----Original Message----- From: Derek Davies [mailto:daviesd2@icrf.icnet.uk] Sent: Wednesday, November 11, 1998 5:00 AM To: cyto-inbox Cc: Cytometry Mailing List Subject: Re: HeLa, K562 & cell cycle... Mark, The standard 70% ethanol fixation procedure followed by PI should give reasonable DNA histograms certainly for K562s. Depending on the HeLa strain that you are using, you may see a broadened G1 peak but it should still be good enough for sorting G1, S, G2. If you dont require live cells afterwards and the fixation doesnt interfere with whatever you have planned subsequently, then this is probably the best place to start. Hoechst is the way to go if you want the cells to be live, but the staining is very much dependent on cell type, you will have to play with the concentrations of H33342 and the time of staining. Usually if there is a broad peak, I would up the concentration - Hoechst can be cytotoxic at high concentrations so be careful not to go too high - I find that 10ug/ml is a good starting point; and if the cells have the MDR phenotype they will be pumping the dye out and the addition of something like verapamil or DiOC5 can help in these situations. You dont say what your Hoechst staining conditions are but I would suggest either leaving the dye on longer or increasing the concentration. Using Hoechst of course has the added advantage that you can exclude dead cells by PI gating so you should be able to see how cytotoxic your staining conditions are. Hope this helps etc, Derek On Tue, 10 Nov 1998, markster wrote: > Do anyone have any experience/comments/references > with regard to staining live, fixed or permeablised > HeLa or K562 cells for sorting G0G1/S/G2M by DNA content > using Hoechst 33342 or PI > I have tried HO342 in 'live' K562 and was not able to sort or > distinguish cell cycle phases. One peak very very broad! > Mark **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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