You can use CFSE on bacteria but you have to ensure that your organism does not actively extrude the dye. Best is to grow them very stationary and use the "used up medium" for washing steps etc. The lower pH will protect the dye from extracellular cleaving and aid the uptake. However, it is still wise to check the pumps. The advantage of the CFSE is that it is an intracellular stain so should give lower interference on the surface of the bugs. However, if they start energising they will pump out unbound dye (leave them deenergised for longer) and once they start dividing they half their fluorescence between daughters (cell tracking studies) so you won't see much after several days. Depending on how you fix them you might modify the surface again. Also consider CCFSE (di-chloro...) as it has a pKa of 4.5 thus is not quenched so much by pH changes in the local environment. The best way to study them inside the cells is to use GFP expression. See Isabelle Hautfort at the Institute of Food Research in Norwich regarding Salmonella http://www.micron.ac.uk/Meetings/Programme2002.pdf or for Staphylococci Phil Hill at Nottingham http://www.nottingham.ac.uk/biosciences/foodsci/academic/phil_hill.html Regards Gerhard Gerhard Nebe-von-Caron Please note change of address 3 The Drive Sharnbrook, Bedfordshire, MK44 1HU, UK Tel: +44 (0)1234 781777 E- mailto:gerne@bigfoot.com -----Original Message----- From: David L Haviland [SMTP:David.L.Haviland@uth.tmc.edu] Sent: Tuesday, June 25, 2002 5:43 PM To: Cytometry Mailing List Subject: Re: FITC-labelling of bacteria Mike: > I have used Molecular Probes Cell Tracker green on viable bacteria for > cell binding experiments using flow for quant. This worked very well > for me. In your hands, how long did the dye remain detectable? I'm thinking of a phagocytosis experiement involving Staph aureus and macrophages, so I need the bugs to be "detectable" for a long time (>6 hour if not days). I need to do some leg work but do you or anyone know if CSFE can be used in bacteria? Many thanks David
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