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I need help on two topics:
1) Fixation of macrophages plus FITC-labelled bacteria samples to assess
phagocytosis in a flow cytometer 4-7 days after fixation. Would fixation
with paraformaldehyde preserve the fluorescence activity for up to 1
week?. Which other fixatives could be used?
2) Quenching of FITC-labelled extracellular bacteria in phagocytosis
experiments. I am trying to quench with ethidium bromide (50mg/ml final
concentration) but in suspensions with only bacterial cells approximately
50% of the cells stain red. The other 50% still stain green colour. Trypan
blue is giving better results, although I might need a washing step prior
to fixation (TB would enter the macrophage cells).
Any comments on how to quench bacterial extracellular FITC fluorescence and
on how to preserve the samples for up to 7 days would be very appreciated.
Thank you very much in advance.
Natalio Garcia Garbi, PhD student
Institute of Aquaculture - Stirling University
Tel: +44.1786.467878 Fax: +44.1786.472133 e-mailngg1@stir.ac.uk
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