Cell Cycle Chromobody® (CCC)

ChromoTek's powerful Cell Cycle Chromobody® enables you to screen compounds such as cancer drugs for effects on the cell cycle and toxicity in one experiment. You can apply this HCA assay in early drug development and validation as well as in basic research.

The Cell Cycle Chromobody® visualizes proliferating cell nuclear antigen (PCNA), the homotrimeric ringshaped protein that encircles the DNA and acts as a stationary loading platform for multiple, transiently interacting factors participating in various DNA transactions. As an essential cellular component, PCNA is highly conserved from yeast to man.

In eukaryotic cells, PCNA plays a key role in DNA replication, repair, cell cycle regulation, and post-replicative transactions like DNA methylation and chromatin remodelling.

Cell Cycle Chromobody® signal during the cell cycle

In G1, the Chromobody® signal is homogeneously distributed throughout the nucleus and cytoplasm. During S phase it accumulates in the nucleus and visualizes the formation of replication foci. In G2, the foci disappear and the cell divides (mitosis).

For simultaneous read out of

  • Cell size and morphology
  • Nuclear morphology
  • Progression of S phase (detailed detection of early-mid-late S phase)
  • Mitosis

Features of Cell Cycle Chromobody

  • Trace dynamic changes during the cell cycle in real time
  • Monitor the distribution of an endogenous cell cycle marker protein - in comparison to fluorescent fusion proteins no overexpression artifact or cytotoxic effects
  • Does not affect cellular functions
  • Available as stable fluorescent HeLa and U2OS cell lines ready to go for cellular screening

Treatment of the U2OS CCC cell line with three exemplary inhibitors of cell cycle progression

16 hrs after addition of increasing concentrations of substances, cells were fixed and DNA counterstained with DAPI. Three independent experiments were performed in duplicate. Image acquisition was performed with an INCell Analyzer 1000 equipped with an 40x air objective. For image analysis and cell classification INCell Workstation software was used to generate a decision tree based on nuclear size, shape and intensity and the amount and size of appearing nuclear granules/ spots (replication foci).

Aphidicolin: a reversible inhibitor of eukaryotic nuclear DNA replication (specific inhibitor of DNA polymerase A,D) blocks the cell cycle at early S phase. With increasing concentrations (0.5, 1, 5, 10, 50, 100 µg/ml), the percentage of cells in G phase declines. In correlation to that the number of cells arrested in early S phase increases. Toxic effects of high Aphidicolin concentrations can also be measured.
Nocodazole: an agent interfering with the polymerization of microtubules. G2-/ M-phase arrest. Cells enter mitosis but can not form metaphase spindles. With increasing concentrations (50, 100, 500 nM, 1, 5, 10 µM), the percentage of cells in G phase declines. In correlation to that the number of cells arrested in mitosis increases. No toxic effects of high Nocodazole concentrations can be detected.
Trichostatin A (TSA): a compound that selectively inhibits the class I and II mammalian histone deacetylase (HDACs). TSA inhibits the eukaryotic cell cycle during mid-/ late-S phase. With increasing concentrations (1, 5, 10, 50, 100, 500 nM), the percentage of cells in G phase declines. In correlation to that the number of cells arrested in progressed S phase increases. No toxic effects of high TSA concentrations can be detected.

Cell Cycle Chromobody® plasmid.

HeLa-Cell Cycle Chromobody®-TagGFP2

U2OS-Cell Cycle Chromobody®-TagGFP2

HeLa-Cell Cycle Chromobody®-TagRFP

U2OS-Cell Cycle Chromobody®-TagRFP

Lentiviral Cell Cycle Chromobody® cell lines

(transduced in collaboration with NMI Tuebingen)

cell linefluorescent protein colour


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