Dressler 1990

Dressler LG. 1990. Controls, standards, and histogram interpretation in DNA flow cytometry. Methods in Cell Biology 33: 157-171.

This author describes and discusses various procedural components and concerns involved in using flow cytometry to measure nuclear DNA contents, in the context of human cancer research and diagnosis.
Among controls, one important control described by this author is the use of cytologic or histologic examination, for example by staining a subsample of a specimen and examining under a compound microscope. In my work, this can be accomplished through Feulgen image analysis densitometry, though I think this author had in mind a less involved technique with the intention of verifying cell types and cell densities. This author also reiterates that the use of internal (ideally co-prepared) standards is critical for flow cytometry.

This paper is divided into procedures for first, fresh and frozen tissues and second, formalin-fixed, paraffin-embedded tissues. For fresh and frozen tissues, the use of duplicates, where one duplicate is the specimen alone, the other the specimen plus the standard, is urged. This fits well with what we have already been doing in our lab, using the unknown specimen alone to determine the approximate range of the position of the peak in the histogram, and the specimen coprepared with a known standard to actually estimate genome size. For fixed tissue, additional procedures are described for removing nuclei from the paraffin matrix, and the DNA index (Vindelov and Christensen 1990) is clearly defined; it is essentially the same as our calculation of genome size by comparison to a known standard.

This author also includes a recipe for freezing medium for use at -70°C, and advice on clarifying peaks in the histogram.