Hydrostatic pressure enhances vital staining with carboxyfluorescein or carboxydichlorofluorescein in Saccharomyces cerevisiae: efficient detection of labeled yeasts by flow cytometry

Abstract

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required D-glucose and was markedly inhibited by 2-deoxy-D-glucose, suggesting that glucose metabolism has a role in the process.

FIG. 1

Pressure-induced accumulation of CF or CDCF in strains IFO2347 (A) and IFO10159 (B). Cells were labeled with 10 μM CFDA or CDCFDA under several hydrostatic pressures for 1 h, and the labeled cells were analyzed by using CytoFluor 2350. Symbols: ○, CF-labeled cells; •, CDCF-labeled cells.

FIG. 2

Histograms of populations of labeled cells. Labeled cells (approximately 100,000) were analyzed by using the Bryte-HS flow cytometry system. (A) Cells of strain IFO2347 subjected to hydrostatic pressures of 0.1, 30, and 60 MPa in the presence of 10 μM CDCFDA for 1 h. (B) Cells of strain IFO10159 subjected to hydrostatic pressures of 0.1, 40, and 60 MPa in the presence of 10 μM CDCFDA for 1 h.

FIG. 3

Effect of hydrostatic pressure on the hydrolysis of CDCFDA in strain IFO2347. Fluorescence was emitted in a hydrostatic chamber with transparent windows and analyzed by using an RF5300PC spectrofluorometer. (A) Hydrolysis of CDCFDA under elevated hydrostatic pressures. A cuvette containing the cells was placed in the chamber, and hydrostatic pressure was applied at time P after addition of 50 μM CDCFDA. (B) Hydrolysis of CDCFDA at atmospheric pressure after preincubation at several hydrostatic pressures. A cuvette containing pressure-adapted cells was placed in the chamber, and then 50 μM CDCFDA was added.

FIG. 4

Effects of d-glucose and 2-deoxyglucose concentrations on the pressure-induced accumulation of CDCF in strain IFO2347. (A) Cells incubated with 10 μM CDCFDA at 0.1 MPa (○) or 30 MPa (•) in the presence of various concentrations of d-glucose in MBA buffer for 1 h. (B) Cells incubated with 10 μM CDCFDA at 0.1 (○) or 30 (•) MPa in the presence of 100 mM d-glucose and various concentrations of 2-deoxyglucose in MBA buffer for 1 h. Labeled cells were analyzed by using CytoFluor 2350.