Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining
2023
Maria V. Kulakova | Eslam S. M. O. Ghazy | Fedor Ryabov | Yaroslav M. Stanishevskiy | Michael O. Agaphonov | Alexander I. Alexandrov
Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in <i>Saccharomyces cerevisiae</i> and <i>Ogataea polymorpha</i> yeasts. The protocol for the DNA content assay was also optimized to be suitable for both <i>Ogataea</i> and <i>Saccharomyces</i> yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.
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