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@stir.ac.uk
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