Data Analysis Pipeline for High Content Screening in Drug Discovery - High-Throughput Image Reconstruction and Analysis - page 211

Figure 9.1 HCS assay formats. (a) Cytoplasmic-nuclear translocation of GFP-Foxo3a. The ratio of
GFP-Foxo3a intensity in the nuclear mask region (circle) and in the cytoplasmic mask region (ring)
was used as the quantitative measurement. (b) Cytometry assay measuring percentage of S-phase
cells. S-phase cells were labeled with Ethynyl-dU incorporation and azido-rhodamine label and all
cells were labeled with Hoechest 33342 dye. (c) Morphology assays on myotube formation and
neurite formation. Myotube length and width, neurite length, and branches, were among the
parameters of interest.

sorting (FACS) is the most common format for cytometry based assays. FACS
requires a large number ( 10 5

of cells and does not provide enough resolution to
score intracellular or morphological changes in cells, whereas HCS based cytometry
requires many fewer (

> )

10 2

cells and can be performed in high spatial resolution,
thus providing higher throughput and information not accessible to conventional
FACS. Cell cycle assay is a cytometry based assay widely adopted in oncology
research where interfering with cell cycle and cell growth is among the main goals
of therapeutic intervention. Cell cycle stages are determined by DNA content, as
well as elevation of other specific cell cycle markers such as phospho-Histone H3
for mitotic cells and cleaved PARP for apoptotic cells [17].

∼

)