Depends what you mean with double labelling. I tend to use goat-anti-mouse*PE (Fab2) from Dako together with carboxyfluorescein and PI in culture models and gam*FITC (Fab2) together with ethidium bromide and propidium iodide in dental plaque samples for the detection of functional cell status combined with antibody differentiation. (see http://www.elsevier.com/homepage/sah/mimet/speciss/1378.pdf from a special issue of http://www.elsevier.com/locate/jmicmeth) If you want to use 2 antibodies you might you might consider premixing them with the secondaries for convenience or even better use isotype specific 2nd step antibodies if your primaries are of different isotype. For real impressive 4-antibody work look at Hutter, K.J. (1992) Simultaneous multiparametric flow cytometric analysis of different species of microorganisms. Monatsschrift Fur Brauwissenschaft 45, 280-284. Identification by immunological methods Cell differentiation by antigen-antibody recognition is the major application of FCM in immunology, where white blood cells are identified by functional epitopes via fluorescent labelled antibodies. Perhaps because the antibody recognition has proven quite successful in the evolution of the immune system, it is the most frequently used bio-recognition assay these days. There are also ever increasing numbers of antibody based ‘rapid tests’ in microbiology like the latex agglutination or the Clearview tests from Oxoid Basingstoke UK, used for culture confirmation or direct testing from for clinical samples. Despite their widespread use in bulk assays and even fluorescent microscopy, there is only a limited amount of literature about bacterial differentiation by antibodies in flow cytometry. A lot of the early publications suffered from poor cluster separation of the antibody labelled event from the background, but Sahar et al (Sahar et al. 1983) already demonstrated the advantage of counterstaining with ethidium bromide to discriminate bacteria from other particles. Others successfully used propidium iodide for that purpose (Tyndall et al. 1985; Donnelly and Baigent, 1986; Völsch et al. 1990). Such counterstaining is not always required, in particular when handling cultured cells which give rise to stronger light scatter signals. Thus in turn they can be used to assay antibodies in serum and the vaccination efficacy in men (Sachsenmeier et al. 1992; Callister et al. 1994; Lim et al. 1994; Callister et al. 1996; Creson et al. 1996; Padilla et al. 1996). Enhanced differentiation by multicolour antibody fluorescence has been demonstrated using separate fluorochromes for two antibodies (McClelland and Pinder, 1994) and four antibodies (Hutter, 1992) simultaneously with a single wavelength excitation. More recent studies by image and flow cytometry have even used combinations of immunofluorescence, rRNA probes (Li et al. 1997) and total DNA labelling (Assmus et al. 1997; Wallner et al. 1997). Regards Gerhard Nebe-von-Caron Research Scientist Applied Science & Technology Group SEAC - Safety and Environmental Assurance Centre Unilever Research, Colworth Laboratory, Sharnbrook, UK - MK44 1LQ Tel: +44 (0)1234 264822, Fax: +44 (0)1234 222552 E- mailto:Gerhard.Nebe-von-Caron@unilever.com -----Original Message----- From: Jane S. Miller [SMTP:miller@medicine.tamu.edu] Sent: Wednesday, October 03, 2001 2:18 PM To: Cytometry Mailing List Subject: dual immunofluorescence of bacteria Has anyone double labeled bacteria for immunofluorescence using the indirect method (primary & secondary antibodies)? I have a client who is trying to do this. Any helpful hints would be appreciated. Jane Miller, Dept. Med. Micro & Immunol., Texas A&M Hlth. Sci. Ctr., College Station, TX
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