Increasing Cytoplasmic fluorescence

Ray Hicks rh208 at cus.cam.ac.uk
Wed Nov 6 08:08:05 EST 1996


Hi,

Is there a chance that a fluorochrome is lingering in your sample uptake
system, and staining your cells when you introduce them to the machine?  If
it's something like PI or AO, it would light up permeablised cells, intact
cells wouldn't stain so quickly, if at all.  I remember (perhaps wrongly)
that the XL "sips" an aliquot from the sample tube, and injects it into the
flow chamber.  This would explain why putting the same tube on at a later
time has the same effect, since the staining would be done in the sample
uptake system, leaving the cells in the tube unaffected.
FS and SS wouldn't be affected, and the effect would probably be
intermittent on a day by day basis, depending on who had used the machine
before you.  The person who introduces the fluorochrome may not have been
doing so much of it when you used your in house permeablisation method etc.

Try running a series of solvent/bleach/detergent through your machine next
time it happens, and see if the effect disappears.  If it does disappear,
find out who's been putting the free dye in and ask them to do the same
when they finish their runs.

Ray


At 8:10 am 5/11/96, Walter Sharp wrote:
>Dear All,
>        Got an unusual problem that we see quite often these days.
>
>When running cytoplasmic markers (TdT, Mu etc) we see the fluorescence
>intensity
>of both the FITC stained cells and non specifically stained controls creep up
>the 4 decade log scale, levelling out after 2 minutes and up by 1 decade.
>Re-running the SAME tube shows the same pattern, starting at baseline and
>levelling off 1 decade up.
>No changes in FS, SS or aquisition rate are evident.
>We never see this with surface staining at all and it doesn't happen with all
>our Leuks.
>The spectral spillover into FL2 (rpe) also shows a concomittant increase.
>We have a four PMT Coulter XL with System II software.
>We currently use Caltag's "Fix and Perm" with Mu and HT6 TdT clone from Dako.
>We did see it once with our previous "in house" permeablisation method but it
>never repeated itself.
>
>Any ideas ?
>
>Wal Sharp
>SQU
>Oman.

                              Ray Hicks
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