We are trying to detect surface markers using the UV line of an Enterprise laser on a coulter altra. We have several unlabeled primary antibodies, and have purchased the Alexa Fluor 350 secondary antibody. Although we see fluorescence via microscopy, we appear unable to detect stained events on the altra. We are using a 488 DL to move light to the PMT and a 488 SP to eliminate side scatter light from the 488 line. Unless I am missing something, this should work but we get no signal from stained cells. We are starting with markers like CD4, where there should be a clear distinction between positive and negative populations. I know these conjugates are primarily designed for microscopy, but I believed that we would easily be able to detect a fluorescence signal this way. The primary and the secondary are species and isotype matched correctly. HELP! Any suggestions, comments or helpful hints would be greatly appreciated. Am I doing something wrong here? -Brian _________________________________ Brian Crucian, Ph.D. (JSC-SD3) NASA-JSC/Wyle Life Sciences Cell and Molecular Research Laboratory 281-483-7061 brian.crucian1@jsc.nasa.gov
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