Howard Mostowski wrote- > Following Alice Givan's and others train of thought on this matter >of one cell vs an aggregate; pardon my naivety on this, could not this >matter be easily settled not by microscope alone, but using the same methods >applied to DNA analysis in discerning 2N vs 4N. A hard copy for all >non-believers and believers alike. An excellent point; a lot of immunofluorescence work is done with no attempt made at doublet discrimination. This demonstrably will cause problems in telling aggregates from dual positives; it causes even more problems when there are not true positives and negatives, as is the case for many activation antigens. In a stimulated culture, there are almost certainly more sticky cells than in an unstimulated one, and the scatter distributions widen, making it difficult to gate out aggregates. Thus, one cannot be sure whether a small population exhibiting higher fluorescence represents cells expressing more of the antigen, aggregates, or both. In most of my work on activation antigens, going back to the early 1980's, I stained everything with Hoechst dye; even in stimulated cultures, there are almost no cells in S, G2, or M phase for 24-30 hr, so it is easy to get rid of the aggregates. With a lot more instruments capable of measuring 6 or more colors, it may be advisable for people to add nuclear stains to their immunofluorescence cocktails to facilitate discrimination of aggregates. -Howard Shapiro
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:38 EST