From: Peter Schroeder (peter.schroeder@medtronic.com)
Date: Mon Apr 20 1998 - 15:11:24 EST
Dear Group,
We have been trying to measure the phagocytosis by isolated monocytes
by using FITC labeled bacteria (Molecular Probes) and quenching the
reaction with trypan blue 70% dye (0.5 to 3 mg/ml, Sigma) to discriminate
between attachment and internalization of bacteria by quenching the FITC
fluorescence of surface bound bacteria leaving the fluorescence of
internalized particles unaltered. All the literature makes this sound very
straight forward but we are having a hard time convincing ourselves that we
are quenching the FITC.
- We can quench the fluorescence of the FITC beads used in the set-up of
the FACsCaliber. (BDIS)
- The FITC labeled bacteria were added to a tube of fixed monocytes (1%
PFA) that had been suspended in a solution of 1mg/ml trypan blue. We then
measured the same mean fluorescence of the monocyte gated region as when we
did not add trypan blue but suspended in 1% PFA. No quenching!
- The fluorescence of just the bacteria in suspension is not changed by
the addition of our trypan blue. No quenching
Is there a trick we are missing..a certain pH, a brand or purity of trypan
blue? The literature also lists ethidium bromide and crystal violet but if
I can make it work I'd rather work with trypan blue.
Thanks For Any Comments.
Best Regards,
Peter Schroeder
Center for Biomaterials Research
Medtronic, Inc.
6800 Shingle Creek Parkway
Brooklyn Center, Minnesota 55430
peter.schroeder@medtronic.com
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