I'd caution against the use of FITC for labeling bacteria especially for any study of bacterial binding. My first project as a post-doc using flow cytometry involved bacterial binding to mammalian cells, and I used FITC-labeled bacteria. In the process, I was able to show that FITC labeling did affect binding (this was based on Scatchard plots of labeled bacteria diluted by unlabled bacteria). Recently we published GFP labeling method that has proved to be very effective (Stapleton AE, Fennell CL, Coder DM, Wobbe CL, Roberts PL, Stamm WE. Precise and rapid assessment of Escherichia coli adherence to vaginal epithelial cells by flow cytometry. Cytometry. 2002 Feb 15;50(1):31-7). Membrane labeling should also be effective though I have not used such for bacteria. If you will be doing lots of studies with a defined set of bacteria, then taking the time to generate GFP-transfected strains would be worth the time; it is a very convenient to have pre-labeled bugs on hand. Dave ======================================== David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com or dcoder1@hotmail.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Michael Herron" <herro001@umn.edu> Sent: Friday, June 14, 2002 4:39 AM Subject: Re: FITC-labelling of bacteria > > 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. > > Hazel Davey wrote: > > > > On 12 Jun 2002 at 9:15, Josef Azem wrote: > > > > > > Dear Flowers! > > > > > > Has anyone have a protocoll for FITC-labelling of bacteria? > > > > Straight FITC rather than FITC labelled antibody? > > > > Here is the method I use: > > > > Stock solution of FITC is 1 mg/ml FITC in acetone. Final > > concentration of FITC incubated with bacteria should be in the 1-10 > > ug range depending on sample / cytometer. Incubation for 15 mins is > > usually sufficient. I think I adapted this from Miller, J.S. and > > Quarles, J.M., 1990. Flow cytometric identification of microorganisms > > by dual staining with FITC and PI. Cytometry 11, 667-675. > > > > As usual you may have to adjust this for your circumstances but this > > should provide a useful stsrting point. > > > > Hazel-- > > Hazel Davey HLR@aber.ac.uk http://pcfcij.dbs.aber.ac.uk/ > > Sefydliad y Gwyddorau Biolegol, Prifysgol Cymru, Aberystwyth > > Inst. Biological Sciences, University of Wales, Aberystwyth > > http://www.nspcc.org.uk/donate-4-free/donate-mainhome.asp > > -- > > ________________________________________________________ > / Michael J. Herron, U of MN, Dept. of Pediatrics/BMT / > / herro001@umn.edu / > / 612-626-4321 Mpls MN 55455 / > /_______________________________________________________/ > >
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