DOI: 10.5281/zenodo.14018551
title: FlowCytometry & CellCycle MtDNA

description: In Saccharomyces cerevisiae, flow cytometry enables semi-quantitative estimation of mitochondrial DNA (mtDNA) levels by measuring the difference in signals between cells lacking mtDNA and those containing it. Using flow cytometry, we investigated mtDNA accumulation in yeast cells under G1 and G2 phase cell cycle arrest conditions, employing thermosensitive mutants cdc4-3 and cdc15-1. Here, we present the source flow cytometry data associated with our manuscript. Cell cycle arrest induced a several-fold increase in mtDNA accumulation in both mutants. The total DNA levels in arrested cells correlated with cell size, suggesting a relationship between individual cell mtDNA quantity and cellular dimensions.
description: Strains and growth medium: In this study we used W303-1A laboratory strain (MATa ade2-101 his3-11 trp1-1 can1-100 leu2-3). We also utilised cdc4-3 thermo-sensitive mutant (Li et al. 2011) and cdc15-2 thermo-sensitive strain(Zyrina et al. 2015). 

Strain Preparation and Cell Cycle Arrest:





Strains cultured overnight at 25°C, inoculated at low density (<2×10^6 cells/ml) to ensure exponential growth phase.




Cell suspensions divided into two pools:a. Control: Fixed in 70% ethanol at 25°C.b. Arrested: Incubated at 37°C for 6 hours, then fixed in 70% ethanol.




OD and cell morphology assessed post-arrest to confirm cell cycle arrest efficiency.



Cell Staining Protocol:





Fixed cells washed 3× with 50 mM sodium citrate buffer (pH 7.0).




RNase A treatment: 1.5 μl (10 mg/ml) in 0.5 ml buffer, 2 hours at 37°C with agitation.




Proteinase K digestion: 5 μl (20 mg/ml in 50% glycerol + TE buffer), 1 hour at 50°C.




Sonication for cell disaggregation.




Fluorophore addition:a. Propidium Iodide (PI): 5 μg/ml final concentration.b. Sytox Green: 0.625 μM final concentration.




Incubation: 4°C for ≥1 hour (PI) or ≥30 minutes (Sytox Green).




Storage: 4°C, light-protected, stable for weeks.



Flow Cytometry Analysis:





Instrument: CytoFlex (Beckman Coulter)




Emission filters:a. PI: PC5.5 (690/50 nm)b. Sytox Green: FITC (525/40 nm)




Data processing: flowCore package (Bioconductor)




Signal transformation: Log2




Peak determination: R multimode package with manual verification



References

Li, Zhijian, Franco J. Vizeacoumar, Sondra Bahr, Jingjing Li, Jonas Warringer, Frederick S. Vizeacoumar, Renqiang Min, et al. 2011. “Systematic Exploration of Essential Yeast Gene Function with Temperature-Sensitive Mutants.” Nature Biotechnology 29 (4): 361–67.

Zyrina, Anna N., Maksim I. Sorokin, Sviatoslav S. Sokolov, Dmitry A. Knorre, and Fedor F. Severin. 2015. “Mitochondrial Retrograde Signaling Inhibits the Survival during Prolong S/G2 Arrest in Saccharomyces Cerevisiae.” Oncotarget 6 (42): 44084–94.
description: Files and variables

All files are flow cytometry data in .fsc format. An event in the .fsc file is a yeast cell, stained with fluorescent intercalating agent (see methods section for more information). We used two types of dyes: Sytox Green and Propidium Iodide. There are only two files for samples stained with Propidium Iodide; the names of these files start with "PI_". All other samples were stained with Sytox Green, and these files are grouped into 3 sets (zip folders). Data in different folders were obtained on different days.

The name of each file contains information about the sample:



The dye (PI - Propidium Iodide or syt - Sytox Green)

Yeast strain (wild type, cdc4, or cdc15)

Presence of mtDNA in this strain (Rho+ or Rho0)

Temperature of incubation (25C - control cells, 37C - arrested cells)