In such a scenario, one can envision that significant changes to chromatin associated with ICP0 can occur prior to our earliest assay time point of 0.5 h.
However, observed changes associated with ICP0 in the mouse model were transient and so the possibility remains that H3K4me2 enrichments of ICP0 may also be transient, occurring prior to the earliest assay time point.
T corresponds to the time point at which the first protein molecule is completed and t to the assay time point.
Furthermore, cell count based on DRAQ5 staining was highly variable and at our assay time point was for most treatments relatively unchanged, thus limiting its usefulness to detect cell death (Fig. 2B ).
Using an automated assay of swimming behavior (Matthies et al. 2006; McDonald et al. 2007), we found that vt25 lacked a fully penetrant Swip phenotype, as evident by its ~50% paralysis in manual scoring and an average ~0.5 Hz thrashing frequency by our final assay time point (10 min) under automated recording.
For all cell-based assays, time points were selected on the basis of studies conducted during the assay development and were considered optimal for the end point being evaluated.
Levels of tumor markers (CA15.3, CEA, and CA27.29) were measured by using a chemiluminometric assay at time points to coincide with radiologic assessments.
Using the laser tweezers assay, the time point of adhesion is tightly controlled and therefore the response in the range of seconds can be analyzed.
After ethanol fixation and propidium iodide staining as described (Detjen et al, 2001), 10 000 cells per assay and time point were analysed by a FACSCalibur Flow Cytometry System (Becton Dickinson, Heidelberg, Germany), using the CellQuest software (Becton Dickinson).
A caspase activation assay was multiplexed with the real-time cell viability assay at multiple time points throughout the time course (Fig. 5 ).
