Dynamic Epitope Expression from Static Cytometry Data: Principles and Reproducibility

摘要: Background An imprecise quantitative sense for the oscillating levels of proteins and their modifications, interactions, translocations as a function cell cycle is fundamentally important cartoon/narrative understanding how works. Mathematical modeling same models would be greatly enhanced by an open-ended methodology providing precise quantification many etc. Here we present that fulfills these features. Methodology Multiparametric flow cytometry was performed on Molt4 cells to measure cyclins A2 B1, phospho-S10-histone H3, DNA content, light scatter (cell size). The resulting 5 dimensional data were analyzed series bivariate plots isolate segments N-dimensional “worm” through space. Sequential, unidirectional regions used assemble expression profiles each parameter frequency. Results Analysis synthesized in which true values where known validated approach. Triplicate experiments demonstrated exceptional reproducibility. Comparison three triplicate stained two methods (single cyclin or dual measurements with common phospho-histone H3 measurements) supported feasibility combining unlimited number epitopes this methodology. sequential degradations followed B1 de-phosphorylation histone precisely mapped. Finally, phase rate during interphase robustly identified. Conclusions Very precise, correlated regulating regulated modifications can produced, limited only available high-quality antibodies. These assembled into large information libraries calibration validation mathematical models.