Solid tumor preparation for flow cytometry using a standard murine model

Abstract

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well‐differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3‐methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol‐water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.