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A method we are currently using, and have luck with in other applications
in which we experience high background fluorescence ( such as
thermal-cycled and FISH samples run on flow ), is the addition of a
counterstain. We use trypan blue, and find that it will quench
intracellular autofluorescence and nonspecific fluorescence thus reducing
your background signal while decreasing your positive signal little if at
all, depending on the marker or probe. ( We have used it with both surface
and internal markers. ) You will also have to experiment some with the
concentration, but we have successfully used a range of 1-10 ug/ml.
Since you are most likely will be quenching intracellular components such
as NADH and flavins you will need to permeabilize the cell to allow the TB
to pass through the membrane. What we typically do is stain with antibody,
then perform a one step fixation and permeabilization step. ( We use 1X
Ortho Permeafix, 50ul per 1-2 million cells, for 1 hour at room
temperature. Fresh (<3 days old) 2% paraformaldehyde for 1 hour on ice
will also work just as well. ) Next, we wash the cells once in 1X PBS, and
then add 500ul of cold trypan blue and incubate for 10 min at 4 degrees.
Spin down, aspirate off excess and resuspend in your analysis buffer.
Trypan blue re-emits this fluorescence in the far red range ( >620 nm ), so
you have to pay attention to your fl2 vs fl3 compensation if you are using
a PE antibody in conjunction with this, but it is easily correctable.
If anyone has any other questions about this technique please write back to
Kbahjat and he will relay the message. We will also be presenting a poster
about this technique at Charleston next week, so feel free to stop by and
chat if you're in the area!
Viki Mosiman
Northwestern Flow Cytometry Core Facility
Northwestern University
Chicago IL
-- Keith Bahjat Northwestern University Kbahjat@nwu.edu
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