Does DAPI stain live and dead cells?
I would be very appreciative if someone introduced me to suitable sources for usual markers (such as DAPI) and its protocol.
Thanks
Hi Masoud,
DAPI is a cell membrane impermeant dye, that will only stain dead cells. In fact it occasionally used to identify dead cells on flow cytometry. If you want a DNA stain that is membrane permeable you can perhaps take Vybrant Dyecycle violet.
I don't know what you mean by sources... Sigma, Life Technologies, millipore? And the protocol: add 1:5000 at the last step of you stain (if you buy the solution).
Hi Masoud,
DAPI is a cell membrane impermeant dye, that will only stain dead cells. In fact it occasionally used to identify dead cells on flow cytometry. If you want a DNA stain that is membrane permeable you can perhaps take Vybrant Dyecycle violet.
I don't know what you mean by sources... Sigma, Life Technologies, millipore? And the protocol: add 1:5000 at the last step of you stain (if you buy the solution).
Thanks to you Hendrik.
I mean information sources such as book chapters, articles, etc in which I can find complete information about these dyes, its protocols and on top of them info about its suitable application in stem cell studies.
DAPI will label any double-stranded DNA it can get to. As noted by Hendrik above DAPI penetrates poorly into most living cells with intact membranes (although some cells will take it up, which has been used as a way to selectively label specific cell populations in some live tissue preparations. For some really nice examples of how that can be used, see some of the A2 amacrine cell work from the labs of DI Vaney, SC Massey, and SL Mills ). In most fixed cell or tissue preparations DAPI will readily penetrate and bind to the Double-stranded DNA. If your preparation has a lot of cells that have died and left debris that includes double stranded DNA, DAPI will label that, too.
The Molecular Probes Handbook is a terrific resource for basic information on all sorts of fluorescent molecules and their applications. (https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook.html?gclid=CMTUtMzr2coCFVQ2aQodv50FHw&s_kwcid=AL!3652!3!81938088071!b!!g!!%2Bmolecular%20%2Bprobes%20%2Bhandbook&ef_id=VrEHCAAAAGRt3Bw6:20160202194408:s)
For what it's worth, Hoechst works great in live cells and fixed cells. You can buy the dye directly and then dilute it, or use NucBlue (google) pre-made solutions. We add it 20 minutes before observation and then wash it out (optional). Very easy, just make sure not to overexpose your cells for live-imaging because the excitation band is near-UV.
Thanks to all. I searched the internet about the protocols in similar areas and find the following site:
http://cshprotocols.cshlp.org/site/misc/subject.xhtml
I think it may be helpful to you in any other areas.
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