Re: Increasing Cytoplasmic fluorescence

H.-G. Kreysch (kreysch@merck.de)
Wed, 06 Nov 1996 10:41:34 +0100

Hi Walter,
The increasing fluorescence you see when analysing fixed and permeabized
cells may result from contamination of the tubings of your FACS with dyes
which stain cytoplasmatic components.
We had similar effects while analysing cells for their cytoplasmatic
cytokines or cytoplasmatic antibodies after having used DNA dyes (PI, 7AAD)
before. Very interesting effects were seen after use of AO.
Try to clean the tubings with 70% ethanol or bleach or replace the tubings.
Good luck
Georg Kreysch

>
>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.
------------------------------------------------------------------------
H.G. Kreysch
Merck KGaA
Biomed Fo/IMO - A25/410 Tel.: (49)6151 - 72 7323
Frankfurter Str. 250 FAX: (49)6151 - 78 1332
D-64271 Darmstadt E-Mail: kreysch@merck.de