Monitoring Cell Cycles

In this issue

Monitoring Cell Cycles
Multicolor Labeling of Cell Nucleus with DNA-Binding Dyes
DAX-J2™ Ratio 580/460, a Ratiometric Nitric Oxide (NO) Probe
Neuraminidase Assay
Detecting Sphingomyelinase Activity
Multiplexing Caspase Activity Detection
Ratio Imaging of Cells with RatioWorks™ Probes
Multicolor Labeling of Dead Cells for Flow Cytometric Analysis
iFluor™ Labeling Probes
iFluor™ Phalloidin Conjugates for Labeling F-actins
trFluor™ Bioconjugates for Developing No Wash ELISA Assays

AssayWise Letters 2013, Vol. 2(2)
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Cell division is just one of several stages that a cell goes through during its lifetime. The cell cycle is a repeating series of events that include growth, DNA synthesis, and cell division. The cell cycle in prokaryotes is quite simple: the cell grows, its DNA replicates, and the cell divides. In eukaryotes, the cell cycle is more complicated. The diagram above represents the cell cycle of a eukaryotic cell. As you can see, the eukaryotic cell cycle has several phases. The mitosis phase (M) actually includes both mitosis and cytokinesis. This is when the nucleus and then the cytoplasm divide. The other three phases (G1, S, and G2) are generally grouped together as an interphase. During the interphase, the cell grows, performs routine life processes, and prepares to divide

The cell cycle has four sequential phases: G0/G1, S, G2, and M. During a cell's passage through cell cycle, its DNA is duplicated in S (synthesis) phase and distributed equally between two daughter cells in M (mitosis) phase. These two phases are separated by two gap phases: G0/G1 and G2. The two gap phases provide time for the cell to grow and double the mass of their proteins and organelles. They are also used by the cells to monitor internal and external conditions before proceeding with the next phase of cell cycle. The cell's passage through cell cycle is controlled by a host of different regulatory proteins.

There are a variety of parameters that can be used for monitoring cell viability and proliferation. In normal cells, DNA density changes depending on whether the cell is growing, dividing, resting or performing its ordinary functions. The progression of the cell cycle is controlled by a complex interplay among various cell cycle regulators. These regulators activate transcription factors, which bind to DNA and turn on or off the production of proteins that result in cell division. Any misstep in this regulatory cascade causes abnormal cell proliferation which underlies many pathological conditions, such as tumor formation. Potential applications for live-cell studies are in the determination of cellular DNA content and cell cycle distribution for detecting variations in growth patterns, for monitoring apoptosis, and for evaluating tumor cell behavior and suppressor gene mechanisms.

Our Cell Meter™ Fluorimetric Cell Cycle Assay Kits are designed to monitor cell cycle progression and proliferation by using our proprietary cell cycle dye in permeabilized and fixed cells. The dye passes through a permeabilized membrane and intercalates into cellular DNA. The signal intensity of the cell cycle dye is directly proportional to DNA content. The percentage of cells in a given sample that are in G0/G1, S and G2/M phases, as well as the cells in the sub-G1 phase prior to apoptosis can be monitored with a flow cytometer.

Left: DNA profile in growing and camptothecin treated Jurkat cells. Jurkat cells were treated without (red) or with 20 µM camptothecin (blue) in a 37 oC, 5% CO2 incubator for about 8 hours, and assayed with Cell Meter™ Fluorimetric Cell Cycle Assay kit. The fluorescence intensity of Nuclear Green™ LCS1 was measured with a FACSCalibur™ flow cytometer using the FL1 channel. In growing Jurkat cells, nuclei stained with Nuclear Green™ LCS1 showed G1, S, and G2 phases (red). In camptothecin treated apoptotic cells (blue), the fluorescence intensity of Nuclear Green™ LCS1 was decreased, and both S and G2 phases were diminished. Right: DNA profile in growing Jurkat cells. Jurkat cells were dye-loaded with Cell Meter™ Fluorimetric Cell Cycle Assay kit and RNase A for 30 minutes. The fluorescence intensity of Nuclear Red™ LCS1 was measured with the FACSCalibur™ (Becton Dickinson, San Jose, CA) flow cytometer using the FL2 channel.

Table 1. Live and fixed cell cycle assays for flow cytometry

Product Name ▲ ▼	Excitation Source (nm) ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit Optimized for 405 nm Violet Laser Excitation	Violet Laser (405 nm)	401	460	100 Tests	22845
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit Green Fluorescence Optimized for Flow Cytometry	Blue Laser (488 nm)	503	527	100 Tests	22841
Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit Red Fluorescence Optimized for Flow Cytometry	Blue Laser (488 nm)	488	615	100 Tests	22860
Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit Red Fluorescence Optimized for Flow Cytometry	Blue/Green Laser (488/532 nm)	537	682	100 Tests	22842