From: Dr. T. D. Prospero (terryprospero@hotmail.com)
Date: Tue Nov 10 1998 - 22:59:50 EST
From: Dr. Terry Prospero terryprospero@hotmail.com ---------- > > Members of the flow cytometry e-mail list, > > I am posting this on behalf of my colleague, who is trying to do three color > flowcytometry analysis on a murine macrophage cell line J774. She is using > Strepavidin Cy-chrome conjugate ( as the third color) for biotinylated anti- > Class I or II MHC antibodies. She gets huge background problem with > Strepavidin Cy-chrome when J774 cells were infected, although she could > titrate resolve the background with 1:6000 dilution when uninfected J774 > cells > are stained. She is desperately looking for help or suggestion from flow > members who have experience with this flourochrome conjugate. > > Sathi > > Her message is here : > ---------------------------------------------------------------------------- > ------------- > I am conducting an in vitro study on Class I and II MHC expression on > macrophages infected with an intracellular pathogen. I am using the murine > macrophage cell line, J774. In my experiments, I have been using > biotinylated anti-mouse Class I and II MHC antibodies in conjugation with a > fluorochrome-conjugated streptavidin (Streptavidin Cy-chrome from > Pharminogen). > > The problem I have encountered is high background with the Streptavidin > Cy-chrome alone with my infected macrophages. I titrated the Streptavidin > Cy-chrome down to 1:6000 with uninfected J774 cells. Background=3%. > However, the background increased dramatically with my infected macrophage > cell line. Background=50% with Streptavidin Cy-chrome alone at 1:6000. If > I dilute the streptavidin any further, the cells will not stain Class I or > II MHC brightly. > > Note: In all the above experiments, I blocked my cells with 10% horse > serum for 2 hrs at 4 degrees Celsius. > > If anyone has suggestions on how I could rectify this problem, please e-mail > with a reply. It would be very much appreciated. > > Thank you in advance, > > > Dr Pizzey's suggestion of Neutravidin may help, if the conjugate is available. A cheap way that may allow you to to keep using your existing streptavidin conjugate is to pre-block the cells. After the biotinylated antibody, include in the first wash some streptavidin pre-mixed with a very small excess of biotin (or you can remove trace free biotin using a PD10 or Pierce Excellulose column just before use) .Non-specific binding sites will be blocked. The free biotin will wash away in the remaining wash steps. Then add your conjugate. I used this approach about ten years ago when I had high backdground problems on a (human) monocyte cell line. There was also a protocol for staining with sequential biotinylated monoclonals published in J Imm Methods in about 1984 (I don't have the ref to hand) where the problem of non-specific binding was resolved by a blocking step. Theintriguing extent of the difference between the control and infected cells would suggest that the pathogen may either itself secrete something to the cell surface; or induce expression of cellular protein(s) - cf. Theileria induction of Tac and Class II - (Dobbelaere et al., Infect. Immun. 58: 3847-3855, 1990) ; or perhaps affect the ER-Golgi secretory system such that an incompletely or incorrectly processed membrane protein precursor appears on the cell surface. The "new" component(s) would be the moieties binding your conjugaet. Thus the possibility exists that there might be something going on of considerable biological significance, rather than solely a troublesome artefact. The worst-case possibility is that the change the pathogen induces results in stickiness for the fluorophore! I don't know of an instance of this. If the blocking doesn't work, try single-staining for your Class II with FITC-streptavidin as a control. Good luck andsuccess to your colleague in her studies Terry Prospero
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