Flow cytometry (ploidy determination, cell cycle analysis, DNA content per nucleus)

During recent years, flow cytometry has established itself as a useful, quick novel method to determine efficiently, reproducibly and at a reduced cost per sample the relative nuclear DNA content and ploidy level of a large number of species (plants and animals), that has also been used to sort cells with different traits from a mixed cell population (e.g. as when separating heterokaryons from parental protoplasts in protoplast fusion experiments for somatic hybrid production) and, more recently, for the analysis of the composition in various chemical components of different tissues (such as apoptotic markers bound to cell compartments). This chapter will focus on the protocols and uses of flow cytometry applied to Medicago truncatula. Basically, a flow cytometer is a fluorescence microscope which analyses moving particles in a suspension. These are excited by a source of light (U.V. or laser) and in turn emit an epifluorescence which is filtered through a series of dichroic mirrors (Figure 1). Then, the inbuilt programme of the equipment converts these signals into a graph plotting the intensity of the epi-fluorescence emitted against the count of cells emitting it at a time given. Thus, a flow cytometer consists of fluidics, optics and electronics, as it measures cells in suspension that flow in single-file through an illuminated volume where they scatter light and emit a fluorescence that is collected, filtered and converted to digital values for storage on a computer (Robinson, 2006).