Cytometric analysis of the cell cycle that includes mitosis has been reported intermittently for many years (see references in1, 2). The most straightforward assays use immunofluorescence of mitotic epitopes combined with DNA content. These epitopes are on single proteins (2, 3), reside on multiple proteins (1, 4), are degraded in mitosis (2, 5), or unmasked during mitosis (6). Although advantages of objectivity, ability to count large numbers of events, and ability to correlate measurements are obvious reasons to use flow or laser scanning cytometry (LSC) to count mitotic cells, the approach has been used infrequently until recently. Now, it is more common to see the use of cytometry to quantify mitotic cells in studies that are biological and not methodological in theme. This may be due to the wide commercial availability of primary conjugates of MPM-2 and phospho-S10-histone H3 antibodies.
An area of cytometry that has seen recent increased interest is the correlated measurement of biochemical activities, especially phosphorylation (7–9). In this work, the central theme, sometimes more implicit than explicit, has been analysis of pathways and networks rather than assays of single activities. Perhaps for the first time, this approach employs cytometry mainly for quantitative correlation and analysis of complexity rather than the more often cited reasons of convenience and objectivity. For analysis of biochemical networks, cytometry is perhaps the most powerful approach available when the questions are directed and limited to ∼20 variables or less. This is because of correlation, precise quantification, and ability to examine all possible states1 within a single or limited number of samples.
Although most emphasis on biochemical networks within the cytometry community is on signaling pathways, the cell cycle is a biochemical network and highly multiparametric approaches to cell cycle analyses can be equally compelling. Indeed, any regulatory pathway can be analyzed by cytometry with powerful results (e.g., 10). The reason for this is that biochemical networks cannot be conceived except dynamically, and cell populations exist in all possible biochemical states with respect to time for a given environment. This is especially easy to see for asynchronous populations of cycling cells in which all biochemical states are extant at any given time. Analysis of the “regulatory” cell cycle within this context is more easily digested when layered on a backbone of phases and stages that we “understand” from kinetics and morphology. In this sense, the backbone cytometry assay is DNA content coupled with a mitotic marker and at least for the late phases (S, G2, and M), cyclins A2 and B1 for somatic cells. For each of these backbone markers, it is good to have alternative markers. For mitosis, there are two reasons not to be satisfied with phospho-S10-histone H3 and MPM-2 as the only cytometric mitotic markers. First, in studies of drug inhibitions and interactions, phospho-S10-histone H3 phosphorylation in mitosis is governed by aurora kinase B (11–13), and therefore, any treatment that results in reduced aurora kinase B activity, may result in a compromised or eliminated backbone. MPM-2 is a good alternative in this case because the epitope is expressed on many proteins and phosphorylated by several enzymes (14, 15). Therefore, in studies in which enzyme activity is depleted, the epitope may not be so depleted so as to render the backbone unusable.2 Second, in multivariate studies matching fluorescence colors to epitopes in terms of fluorophore characteristics (wavelength, extinction coefficient, and quantum yield) and antigen abundance is a continuous challenge (for a discussion of similar problems on surface immunophenotype, see 17). While many phosphorylated epitopes, which turn out to be the best mitotic markers, are abundant in mitosis, the commercial availability of only two fluorochromes is limiting. Therefore, it would be valuable to have additional antibodies to robust markers available in forms that can be integrated in assays of several mouse monoclonal antibodies. The serine at residue 780 of Rb is a specific substrate of cyclin D/Cdk4 (18, 19) and not Cdk2. We discovered by accident that antibody raised to the phospho-peptide, RPPTLS780PIPHIPR stains mitotic cells intensely. Here, we show that the epitope, defined by this antibody is a robust mitotic marker that is equivalent to MPM-2.