Optimization of yeast cell cycle analysis and morphological characterization by multispectral imaging flow cytometry

Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
Cytometry Part A (Impact Factor: 2.93). 09/2008; 73(9):825-33. DOI: 10.1002/cyto.a.20609
Source: PubMed


Budding yeast Saccharoymyces cerevisiae is a powerful model system for analyzing eukaryotic cell cycle regulation. Yeast cell cycle analysis is typically performed by visual analysis or flow cytometry, and both have limitations in the scope and accuracy of data obtained. This study demonstrates how multispectral imaging flow cytometry (MIFC) provides precise quantitation of cell cycle distribution and morphological phenotypes of yeast cells in flow. Cell cycle analysis of wild-type yeast, nap1Delta, and yeast overexpressing NAP1, was performed visually, by flow cytometry and by MIFC. Quantitative morphological analysis employed measurements of cellular length, thickness, and aspect ratio in an algorithm to calculate a novel feature, bud length. MIFC demonstrated reliable quantification of the yeast cell cycle compared to morphological and flow cytometric analyses. By employing this technique, we observed both the G2/M delay and elongated buds previously described in the nap1Delta strain. Using MIFC, we demonstrate that overexpression of NAP1 causes elongated buds yet only a minor disruption in the cell cycle. The different effects of NAP1 expression level on cell cycle and morphology suggests that these phenotypes are independent. Unlike conventional yeast flow cytometry, MIFC generates complete cell cycle profiles and concurrently offers multiple parameters for morphological analysis.

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    • "To make a distinction between G2 and S-phase cells within populations having a 2N DNA content, we used multispectral imaging flow cytometry according to a previously published protocol [71], but with the following changes. Sytox-green-stained cells were resuspended in phosphate-buffered saline at a concentration of 107 cells/ml prior to loading them into an Imagestream imaging flow cytometer (Amnis Corporation). "
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