Automated Cell Synchrony Determination Using Microscopic Imaging In Live Cells

By Xavier Amouretti, Peter Banks, Brad Larson, and Paul Held, BioTek Instruments

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.