Macrophage autofluorescence - Problems with autofluorescence (Oct/23/2003 )

Hi! I was just wondering if anyone has ever had the problem of autofluorescence coming off of murine macrophages. I'm working with the J774A.1 murine macrophage cell line and I'm infecting them with a bacteria that survives intracellularly. We're screening a library containing GFP as a reporter in the bacteria in this fashion but the problem seems like there is a low level of autofluorescence coming off of the macrophages which is overpowering the fluorescence coming out of the library during infection. The library has a very low expression of GFP and it seems as though the background fluorescence from the mac is only at a slightly lower level than this when viewed on the scope. Does anyone have any suggestions as to how I can get around this problem, possibly by quenching the background fluorescence somehow? Thanks!

You can quench the autofluorescence by using trypanblue or ethidiumbromide - but I´m not sure if this has any influence on GFP fluorescence...

I work with J774 macrophages and I infect them with Mycobacteria that express GFP. I have never had anyt autofluorescence cumming off the macrophage! Im wondering if your microscopy is correct and you are using the correct filter cubes etc?