Re: PI Staining

Ryan Hung (rhung@vcn.bc.ca)
Thu, 10 Jul 1997 15:18:28 -0700 (PDT)

On Wed, 9 Jul 1997, John Ladasky wrote:
> Unlike monoclonal antibodies, PI has a fairly high off rate. So
> having excess stain in the solution helps to keep all of the binding sites
> of the DNA saturated, thus facilitating DNA quantitation. If you just want
> to distinguish live cells from dead cells, you can probably get away with
> washing away the excess PI. But not for long -- after a few hours the dead
> cells would not fluoresce significantly more than the live ones.

Hmm, is this true for unwashed cells as well? I've been using a one-step
hypotonic lysing buffer that contains PI (50 ug/ml), but I've been varying
the time (up to several hours) before reading on the FACS scanner. As
such, would the PI saturate all nuclei, giving a reduced amount of
hypodiploid fluoresence and sending everything up to the G0/G1 peak (thus
giving abnormally low levels of apoptosis)?

Ryan.

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