Simple detection method for distinguishing dead and living yeast colonies

Kucsera, Judit; Yarita, Kyoko; Takeo, Kanji

doi:10.1016/s0167-7012(00)00136-6

Cited by 75 publications

(58 citation statements)

References 7 publications

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“…Cell viability was evaluated by Trypan blue staining (50) and by a commercial live/dead yeast viability assay (Invitrogen) (47,51). Trypan blue is a diazo dye that stains only dead cells; live cells with intact cell membranes are not stained.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast

Poulsen

Fenyvuesvolgyi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The Zika virus (ZIKV) causes microcephaly and the Guillain-Barré syndrome. Little is known about how ZIKV causes these conditions or which ZIKV viral protein(s) is responsible for the associated ZIKVinduced cytopathic effects, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. We used fission yeast for the rapid, global functional analysis of the ZIKV genome. All 14 proteins or small peptides were produced under an inducible promoter, and we measured the intracellular localization and the specific effects on ZIKV-associated cytopathic activities of each protein. The subcellular localization of each ZIKV protein was in overall agreement with its predicted protein structure. Five structural and two nonstructural ZIKV proteins showed various levels of cytopathic effects. The expression of these ZIKV proteins restricted cell proliferation, induced hypertrophy, or triggered cellular oxidative stress leading to cell death. The expression of premembrane protein (prM) resulted in cell-cycle G1 accumulation, whereas membrane-anchored capsid (anaC), membrane protein (M), envelope protein (E), and nonstructural protein 4A (NS4A) caused cell-cycle G2/M accumulation. A mechanistic study revealed that NS4A-induced cellular hypertrophy and growth restriction were mediated specifically through the target of rapamycin (TOR) cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41. These findings should provide a reference for future research on the prevention and treatment of ZIKV diseases. T he recent Zika virus (ZIKV) outbreak was surprising in its rapidity and alarming in its association with microcephaly in newborns (1-3) and the Guillain-Barré syndrome in adults (4, 5). Currently, little is known about ZIKV pathogenicity. ZIKV infects various neuronal cells and affects brain neurogenesis by reducing cellular proliferation and inducing cell-cycle abnormalities, cell death/apoptosis, and autophagy (2, 3, 6-8). However, it is not known which viral determinant(s) cause these cytopathic effects. The objective of this study was to identify ZIKV factors responsible for the ZIKV-mediated cytopathic effects that underlie the diseases associated with ZIKV.

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Section: Methodsmentioning

confidence: 99%

“…We tested whether any of the seven ZIKV proteins tested above would cause cell death. Cell death was first evaluated by staining cells expressing ZIKV protein with a vital diazo dye, Trypan blue (50). As shown in Fig.…”

Section: Zikv Proteins Cause Nuclear Fragmentation and Cell-cycle Dysmentioning

confidence: 99%

Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast

Poulsen

Fenyvuesvolgyi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Both white and opaque starter cultures were grown aerobically for 24 h at 25°C in 5 ml of SC medium. After growth, the cells were washed three times in PBS, resuspended at a cell concentration of 1.2 ϫ 10 7 cells/ml in 5 ml of fresh SC medium supplemented with three different concentrations (2,20, and 40 M) of farnesol from a stock solution of 10 mM farnesol in methanol, and shaken (250 rpm) at 25°C for 4 h. Cells were assessed for viability by Evans blue (13) and propidium iodide (27) staining after every hour. In each case, 1 ml of cell culture was harvested, washed in PBS three times, and stained with either 1 mg/ml Evans blue or 50 g/ml propidium iodide for 5 min.…”

Section: Methodsmentioning

confidence: 99%

In Vivo and In Vitro Anaerobic Mating in Candida albicans

et al. 2007

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Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.

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“…The culture was grown to the stationary phase for 18 h at 25°C in a shaking water bath. The blastoconidia were collected, washed with phosphate-buffered saline (PBS), and enumerated using a hemacytometer (Reichert, Buffalo, N.Y.) and trypan blue dye exclusion (24). Viable cells were adjusted to 10 8 C. albicans cells/ml and used to infect gingival epithelial cell cultures.…”

Section: Methodsmentioning

confidence: 99%

“…To quantify the number of C. albicans cells adhering to the monolayer and entering the cells, infected cultures were washed six times with Hanks' balanced salts solution. The cells then were lysed with 1% (vol/vol) Triton X-100 in PBS, and C. albicans viability and numbers were estimated by trypan blue dye exclusion as described previously (24). This assay quantifies the total number of fungus cells bound to the outside of and internalized by the epithelial cells.…”

Section: Methodsmentioning

confidence: 99%