 | Cell proliferation and cell cycle analysis play critical roles in drug
discovery. Modulation of cell division has implications for a broad
variety of target processes e.g. oncogenesis, angiogenesis,
apoptosis and inflammation. Cellular proliferation also represents a
sensitive marker of cytotoxicity. High Content Screening (HCS) is a
powerful tool for detailed investigation of cellular responses to
pharmacological compounds. Millipore has developed a panel of
platform-independent HCS assays for cell cycle analysis. Each is
immunofluorescence-based, utilizing high quality reagents and
validated protocols. Extensively characterized primary antibodies
against key cell cycle markers phospho-histone H3, Ki-67, cyclin B1
and BrdU are employed in combination with FITC- or Cy3-
conjugated secondary antibodies and a nuclear stain. The assays
permit flexibility with regard to primary antibody species, secondary
antibody fluorophore, and the choice of single or multiplexed
immunolabeling, for fine discrimination of compound effects on cell
cycle. We present cell cycle profiling data using human HeLa and
A549 cells demonstrating the high signal quality achieved with
these reagents, and provide dose responses for cell cycle
modulating drugs, including vinblastine, etoposide, aphidicolin and
paclitaxel. We use the assays to calculate mitotic and proliferation
indices, and to discriminate between cell populations at various
phases of the cell cycle. Our data also indicate AC50 values for
compounds causing cytotoxicity. Additionally, we show that at
working concentrations the reagents exhibit stability for >24 hours
at room temperature, offering a great benefit for large-scale
screening applications. These assays represent a simple, ready-touse,
validated strategy for characterization of agents which
modulate the cell cycle, for cancer drug screening, aurora kinase
profiling, and for in vitro toxicology studies - representing broad
potential for drug screening and development.
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