Hi have you tried autofluorescence compensation ?: Alberti S, Parks DR, Herzenberg LA.A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry. 1987 Mar;8(2):114-9. Roederer M, Murphy RF.Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry. 1986 Nov;7(6):558-65. do you know the transfection efficiency of the protoplasts ? and why are there 2 populations that differ in FL1? Best wishes Rachel ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: Ibtissam Abdul-Jabbar > Sent: Wednesday, June 5, 2002 10:12 PM > To: Cytometry Mailing List > Subject: Auto-fluorescence > > <<File: autofluorescence.doc>> > 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. > <<autofluorescence.doc>> > many thanks > yours > iaj > please use the above metioned email address. > >
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