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Automated Cell Synchrony Determination Using Microscopic Imaging in Live Cells


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Authors: Xavier Amouretti, Peter Banks, Brad Larson, and Paul Held, BioTek Instruments, Inc.

The replication of cells is responsible for tissue, organ, and species growth and reproduction. Proliferating cells repeatedly transition between cellular duplication or interphase and cell division or mitosis. Progression through the cell cycle is a highly regulated process with a number of checkpoints. Likewise, the effect of compounds on cellular proliferation is a key element of the ADME/Tox drug discovery process. For these reasons the assessment of cell cycle progression under experimental conditions is highly desirable. With nuclear staining, cells can be identified as being in G1 or G2/M based on the intensity of nuclear fluorescence. Cells in G2/M having approximately 2X the fluorescence intensity as cells in G1. Treatment of cells by compounds can result in cells being halted at various points of the cell cycle. For example, exposure to increasing concentrations of nocodazole results in a greater percentage of cells having a 2X nuclear content as measured by nuclear staining intensity. At high concentrations approximately 80% of the cell population have 2X nuclear content; whereas at low concentration or with untreated cell, the cell sub-population is about 15%. Vinblastine treatment of cultured cells has also been shown to stall cells in G2 phase of the cell cycle. With increasing concentrations of vinblastine the percentage of cells identified as G2/M by nuclear staining increases 3-fold, while cells identified as having G1 content decreases accordingly. Conversely, hydroxyurea, which halts cells in S-phase, reduces the fraction of G2 cells in NIH3T3 cells in a concentration dependant manner. Untreated NIH3T3 cells have approximately 15% of their numbers in G2 phase of the cell cycle; with high concentrations of hydroxyurea this percentage falls to about 1% after a 24 hours exposure.

Cellular nuclear content has also be assessed using frequency histograms of fluorescence intensity. Untreated asynchronous HeLa cells grown in 10% serum demonstrate a major frequency peak that corresponds to cells with G1 nuclear content, as well as a smaller less well defined peak that corresponds to G2. The intermediate region of nuclear content between G1 and G2 cells represents cells in S-phase undergoing the process of DNA duplication. Cells treated with thymidine demonstrate a build-up of cells stalled in S phase.

Cells actively duplicating their DNA (S-phase) can be identified using incorporation of EdU into cellular DNA with subsequent labeling with fluorescent tags via Click-iT® chemistry. Cells in the presence of full serum or newly released from a G1 cell cycle block exhibit marked increases in labeled nuclei as compared to cells serum starved.

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