The long-range objective is to create a robust, precise, and accurate model of the mammalian cell cycle based on N dimensional clustering of cytometric measurements of the activity of rate limiting molecules for cell cycle traverse. At present, this is encapsulated by an intermediate Transition State Model of Mitosis that is based on measurements of DNA content, two morphological parameters (DNA pulse shape and cell size), the expression of cyclin A, cyclin B1, and phospho-serine 10 histone H3. In mitosis, quantification of cyclin A is a measurement of the activity of the Anaphase Promoting Complex (APC) in conjunction with Cdc20; Cyclin B1 reads out the activity of the APC in conjunction with Cdhl. Phospho-S10-histone H3 immunoreactivity reports the activity of Aurora B kinase. For human cells lines, the conjunction of these measurements results in the ability to quantify the frequency of multi-dimensional clusters with sharply defined boundaries that represent transition states that closely approximate morphological mitosis i.e., post- G2 -> prophase --> prometaphase -> metaphase --) anaphase/telophase --> cytokinesis --) new born GI. Because these clusters are frequencies of cells residing in each transition state, the size of the cluster is proportional to the time the analyte population existed in each state at sampling time. Within this analysis, but dependent on fewer parameters, the remainder of the cell cycle (G1, S, G2) can be quantified. The specific aims of this proposal are: (1) to enhance the current analysis by adding 2 parameters aimed at simultaneous quantification of GO and apoptotic cells, and add a third variable parameter; (2) develop a primary set of cytometry-grade cell cycle probes with a uniform and low Kd with the yeast display system. These include phospho-epitopes of substrates of Nek2, PIk-1, cyclin Bl/Cdkl, and Aurora B kinase. (3a) determine the relationship between classical mitosis and cytometrically-defined cell state clusters. (3b) test the hypothesis that the conjunction of phospho-cyclin B1 measurements and other mitotic enzyme activity measurements define unique, novel cell states. (3c) test the hypothesis that the Transition State model can be generalized through GI.