Vindeløv and Christensen 1990

Vindeløv L, Christensen IJ. 1990. An integrated set of methods for routine flow cytometric DNA analysis. Methods in Cell Biology 33: 127-137.

These authors present a set of protocols for standard flow cytometry in a clinical setting. The variables that can be obtained from flow cytometry are the number of subpopulations of cells with different DNA contents in a specimen, the relative sizes of these subpopulations, the “DNA index (DI)”, and the fractions of cells in G1, S, and G2 + M phases. DNA index is used here in a human-cancer context, and is roughly equivalent to nuclear DNA content per nucleus. In running flow cytometry, there are six major problems to overcome. These are sample acquisition, storage, standardization, staining, flow cytometry itself, and statistical analysis, also referred to here as “deconvolution”.

Optimal methods for sample acquisition vary with specimen type; these authors were apparently concerned primarily with the differences between soft and solid tumours from humans, but the point applies across eukaryotes in my opinion. These authors recommend storage of specimens at -80°C with DMSO, but caution that specimens should be frozen and thawed at most once. I am not certain that DMSO is a good idea. Standardization is achieved through the use of internal standards; these authors recommend erythrocytes (blood) from chicken (Gallus gallus domesticus) and trout (Onchorynchus mykiss). These authors describe a staining protocol in three steps, that includes the use of trypsin during cell dissociation and a trypsin inhibitor in a later step to prevent interaction with Propidium iodide (see also Krishan 1990). The chicken blood is described as being mixed with heparin at the time of collection, but no mention is made here of the potential interactions between heparin and Propidium iodide (Krishan 1990).

During flow cytometry, the positions of the peaks in the histogram corresponding to the standards (chicken and trout) are used to determine the DNA index of the measured specimen. These histogram heights can be made equal by mixing the standards together and with the specimen in a manner described by these authors; essentially, cell concentrations are measured and the known fluorescence characteristics of the standards are taken into account. Agitation of specimens will tend to increase the presence of debris and the occurrence of nuclear clumping.

These authors report satisfactory results from these protocols with 17 000 samples for all (human) tissues except sperm.