Phagocytosis - FITC quenching

Natalio Garcia ngg1 at stir.ac.uk
Mon Feb 10 11:16:37 EST 1997


Hello,

We have a problem I hope someone can throw a bit of light into.

We are trying to monitor phagocytosis of FITC labelled bacteria using
rainbow trout phagocytes and Micrococcus luteus, Aeromonas hydrophila and
Yersinia ruckeri as the bacterial targets. The quenching of FITC bound to
extracellular bacteria is attempted with the Quenching solution from
Phagotest kit (Orpegen Pharma). This works well with FITC-E. coli, also
supplied in the kit. However we are not interested in studying the
phagocytosis of this particular sp.

The problem is that, under the conditions used, this quenching solution is
not effective in removing the FITC fluorescence from neither M. luteus,
A.hydrophila nor Y. ruckeri.

We do not know enough to ascertain wether the failure in the quenching of
extracellular fluorescence is due to the specificity of the Quenching
solution for E. coli or wether it is due to the FITC labelling conditions
we apply. Two different buffers for FITC labelling  have been tried (PBS pH
7.4 and carbonate-bicarbonate pH 9.6), both unsuccesfully.

The conditions under which bacterial cells are labelled with FITC are:
growing bacterial cells in BHI broth for 18h at 22°C, washing and labelling
with FITC (1, 0.5 or 0.1 mg/ml final concentration) for 60 min. After the
phagocytosis is allowed, the Quenching solution is applied following
manufacturer's instructions.


If someone has any experience with this I would be very grateful for any
comments.

A summary of suggestions will be posted to the list.


Thank you in advance.

Natalio Garcia Garbi, PhD student
Institute of Aquaculture
Stirling University - Scotland FK9 4LA (UK)
Tel: +44.1786.467878  Fax: +44.1786.472133  e-mail: ngg1 at stir.ac.uk





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