while scattered laser light is crude and rude relative to fluorescence measurements, the use of a forward scatter pulse-width gate helps reduce the number of doublets and aggregates. Using such a gate significantly improves sort purities. td Howard Shapiro wrote: > 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
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