iajabbar at cicr.uq.edu.au
Wed Jun 5 21:12:31 EST 2002
Dear experts, it is the old and reoccurring problem. I have to compare the expression of GFP protein in protoplasts transfected with plasmids carrying the GFP modified gene, In addition to the difficulty in standardizing the method I am facing the huge difficulty in distinguishing and minimizing the auto fluorescence signal in the FL1 region. cells are big, 30ul in diameter at least, and they get bigger after transfection so the real negative is transfected with or without the tested plasmid. Using the 70 Nozzle and triggering on SSC machine (MoFlo) on SSC using log value for the FSC and FL1,
the question is there any way to treat the cell with crystal violate or trypan blue to reduce the auto fluorescence. other option to gated the data in differently (please see attach for the gating strategy I am using), to use other filters, or to stain the cell with anti-GFP-FITC to enhances the differences. I will deeply appreciate your input in this meter.
please use the above metioned email address.
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