[A Modified Procedure to Isolate Synchronous Cells from Yeasts with Continuous Percoll Density Gradient and Their...

Area of Science:

• Cell Biology
• Biophysics
• Spectroscopy

Background:

• Synchronous cell cultures are crucial for studying cell cycle dynamics.
• Isolating quiescent (G0) yeast cells from asynchronous cultures presents technical challenges.
• Previous methods for yeast cell fractionation lack efficiency and specificity.

Purpose of the Study:

• To develop an efficient and economical method for isolating synchronous G0 yeast cells.
• To characterize the isolated G0 cells using microscopy and Raman spectroscopy.
• To differentiate between synchronous G0 and asynchronous yeast cells at a single-cell level.

Main Methods:

• Modified Percoll density gradient centrifugation for cell fractionation.
• Differential interference contrast (DIC) and phase contrast microscopy for morphological analysis.
• Single-cell Raman spectroscopy and Principal Component Analysis (PCA) for chemical composition analysis.

Main Results:

• The modified Percoll method effectively separated yeast cells into two distinct fractions.
• The lower fraction consisted of unbudded, unicellular quiescent G0 yeast cells with high refractive properties and glycogen content.
• Raman spectroscopy revealed higher concentrations of proteins, carbohydrates, and nucleic acids in G0 cells compared to non-G0 cells.
• PCA demonstrated homogeneity within G0 cell populations and heterogeneity within asynchronous populations.

Conclusions:

• The modified Percoll density gradient centrifugation is a feasible, economical, and efficient method for isolating synchronous G0 yeast cells.
• Single-cell Raman spectroscopy combined with PCA can reliably distinguish between synchronous G0 and asynchronous yeast cells.
• The findings provide a valuable tool for yeast cell cycle research and understanding cellular quiescence.