Well, having gotten propidium iodide to finally work, I'm embarking on TdT staining to assay for apoptosis, as I'm trying to do dual TdT and surface antibody staining to identify apoptosis in heterogeneous populations of cells (human PBMC). BTW, thanks to the mailing list for all the help on PI staining--the secret turned out to be knowing what to look for. To those who are still having trouble with the technique, make sure you're using FSC and SSC settings for nuclei rather than for whole cells. I initially set FSC and SSC parameters using whole cells as control samples, then when I fed the PI-stained sample through, I got nothing, as they were nuclei suspensions. Now, with the TdT assay, I've been running into difficulties determining where the apoptosis cells actually are. I'm using the Boehringer Mannheim "In Situ Cell Death Detection Kit, Fluorescein". However, the pamphlet is not at all clear on how to analyze the data. What I have observed so far is this: with negative control cells displayed on a logarithmic histogram of FL-1, I get one large peak at around 10^1. With positive control (camptothecin-treated) cells, that large peak is shifted slightly to the left. Furthermore, the number of cells with high dUTP incorporation increases. My difficulty in interpretation lies in this: which is the significant property of the data? The shift in the large peak to the right, or the increase in high dUTP incorporation? In literature, either one or the other seems to be used to evaluate degree of apoptosis. Does anybody have any suggestions about interpretation of data from the TdT assay? Thanks, Ryan. _/ \__/ \__/ \__/ \__/ \__/ \__/ \__/rhung@vcn.bc.ca__/ \__/ \__/ \_Apoptosis=programmed cell death/ \__/ \rwhung@unixg.ubc.ca_/ \__/ \__ _/ --you can't live without it!/ \__/ \http://www.vcn.bc.ca/people/rhung \__/ \__/ \__/ \__/ \__/ \__/ \__/ \My words Copyright (C) 1997 \__
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