Cell Cycle and Proliferation
Cell Cycle and Proliferation
Last Updated on Saturday, 18 February 2012 19:36
Overview
High-content enables the quantification of cell proliferation activity by measuring DNA content, the state of cell cycle-associated proteins, and morphological and structural changes in individual cells and in cell populations. Multi-parameter readouts of targets leverages the power of high content and allows multiple cell cycle states to be investigated in conjunction with other markers.
At any given moment during development from a stem cell to the fully differentiated state, a normal cell is either dividing (mitosis) or not (interphase). The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. The cell cycle can be divided into four different phases, G1, S, G2 and M. If cells become quiescent, the cells are in the G0 phase.
The cell cycle can be analyzed by measuring DNA content and by detecting and correlating cell cycle-specific intracellular targets. For example, measuring the incorporation of BrdU is used to detect DNA replication in the S phase, while Ki67 is expressed during G1, S, G2 and M phases but is absent in the G0 phase. Other examples include phospho-Histone H3 (M phase marker), cyclins/Cdks, Phospho-Rb and PLK. Aberrations in cell cycle control can lead to cancer or cell death. High content assays uisng the Thermo Scientific HCS Platforms are ideal for quantification of cell proliferation activity by simultaneously measuring and correlating multiple parameters relating to the cell cycle.
Application Example
Application Example: Cell Cycle and Proliferation
Cell stress, cell proliferation and the characterization of agents that either promote or inhibit cell proliferation are particularly of interest in cell biology and drug discovery research. One method to measure cell proliferation relies on BrdU incorporation into DNA, followed by indirect immunofluorescence detection, where cells are imaged and analyzed with fluorescence microscopy. The Cellomics BrdU Cell Proliferation Assay Reagents have been specifically designed to enable BrdU detection in nuclear DNA with gentle cell treatments and a quick, efficient labeling procedure.The procedure also enables multiplexed detection of BrdU with antibodies towards other cellular targets. The DNA binding dye DAPI is used to determine the cell cycle phases by DNA content.
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| Images of A549 and A431 cells stained with Thermo Scientific Cellomics Cell Proliferation Reagents. Cells were seeded at 5,000 cells per well on 96-well microplates and treated with the drugs for 2 hours at 37°C, 5% CO2 in a humidified incubator, then cells were incubated with 40 μM BrdU for 30 minutes. Cell nuclei were labeled with DAPI. The DNA content, BrdU and Ki67 intensities were quantitatively analyzed on the ArrayScan VTI HCS Reader. Cell cycle inhibitory drugs, aphidicolin (1 μg/ml) and hydroxyurea (100 mM) efficiently block BrdU incorporation without changes in Ki67 level. (Inset: DNA content histogram from ArrayScan VTI; DAPI-blue; BrdU-green; Ki67-red). |
