Increasing Cytoplasmic fluorescence

Pizzo,Eugene Pizzo at
Sat Nov 9 10:04:34 EST 1996

PI staining is very susceptible to variation from buffer tonicity and unless
your sample buffer is identical in tonicity to the sheath fluid running in 
machine you may see this equilibration phenomena.  Although the machine
is designed to avoid this - laminar flow - there is a small backflush each 
you change samples.

Gene Pizzo UCONN Health
From: Hector Nolla
To: cyto-inbox
Cc: Cytometry Mailing List
Subject: Re: Increasing Cytoplasmic fluorescence
Date: Thursday, November 07, 1996 8:19AM

Dear Walter,
This may be only a coincidence but what you described is similar to the
"intracellular" fluorescence intensity drift that we sometimes get from
DNA stains like Hoechst and PI. In our case, fluorescence intensity can
either go up or down(most of the time it goes up), skewing the
fluorescence peak and thus increasing CV's, which can be a real "bummer"
if you intent to do cell cycle analysis. This fluorescence intensity drift
can be reduced if we let the sample equilibrate in the sample tubing(by
either running the sample for 30 seconds to 1 min, or by stopping sample
flow thus allowing the sample to remain in the sample tubing).

My best definition for this phenomena is "sample tubing equilibration",
however, this is only "hearsay". Maybe someone out there could shed some
"specific light" as to why this happens? Thanks.

Hector Nolla
Department of Oceanography
University of Hawaii

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