The consensus is that LacZ/FDG wis incompatable with the permeablization step
required for cytoplasmic staining. The solutions to this problem appear to
include: (1)GFP as the reporter gener, with a PE-Ab (keeping in mind that
PE cytoplasmic staining can be more difficult than similar staining with
FITC); (2) Use of an anti-bGal antibody to detect the presence of the
reorter gene product.
Original responses, including a protocol are below. Thanks, everyone!
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GFP will not leak out but the fluorescien product from FDG hydrolysis
definately will. This should be a workable system with a little
experimentation.
Thomas Delohery |Internet: t-delohery@ski.mskcc.org
I only have experience using GFP (eGFP from Clonentech), and for us the
standard 2% PFA, 0.05% Saponin didn't destroy our GFP Staining, and GFP
doesn't leak out of the cells. Haven't tried intra cellular staining on the
same time, could be tricky of course because GFP = green fluorescence, so
you would have to use PE or a conjugate for the intracellular staining..
although this works in some cases, background could be higher than with
FITC...
Sorry that I can't help you more, but maybe others have more experience in
this...
Matthias Haury, mhaury@pasteur.fr
I do cytoplasmic cytokine staining with lymphocytes expressing GFP
(not a fusion protein, but a plain GFP, S65T with humanized codons),
and it works fine.
1. Fix cells in 4 % paraformaldehyde/PBS at 37 oC for 5 min. (I don't
know if this is the best fixation. I have never tried different
conditions because I got a reasonable result with this.)
2. Wash once or twice with PBS/1% FCS/0.1 % sodium azide ("FACS
staining buffer).
3. Suspend the cells in PBS/1% FCS/0.1 % sodium azide/0.1 % saponin
("Permeabilization buffer"), incubate 5 min on ice, and spin.
4. Remove supernatant and stain with antibody diluted in
permeabilization buffer.
5. Wash with permiabilization buffer.
6. Resuspend in FACS staining buffer and analyze.
It is always better to use fresh PFA solution. Soluble GFP needs to be
fixed in place before permeabilization. So I don't think you can
follow the reporter activity in a live cell, if that's what you are
trying to do.
Mayumi Naramura, M.D., e-mail:mnaramura@atlas.niaid.nih.gov
Permeabilization will probably completely leak the FDG stain out. GFP should
stay in; you just need to find fixation conditions that don't destroy its
fluorescence. The other option you have for bGal is to use antibody
staining--there are antibodies to bGal which work with intracellular staining.
Obviously, not as sensitive as FDG (by couple of orders of magnitude), but
should be roughly equivalent to GFP.
Mario Roederer <Roederer@Beadle.Stanford.EDU>
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