Use of flow-cytometry to distinguish between haploid and diploid strains of Aspergillus fumigatus

Lucas, J. Ramón De; Domínguez, Ana I.; Mendoza, Alma; Laborda, F.

doi:10.4148/1941-4765.1249

Cited by 4 publications

(7 citation statements)

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“…RNAse A concentrations and incubation times broadly vary among FCM studies of fungi. Concentrations fluctuate approximately around 1 mg/mL , and incubation times largely range from 1 h to overnight . Our results (see “Methods of Optimization Steps”) indicate that the concentration of 0.1 mg/mL and incubation time of 15 min is sufficient for Geosmithia species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Our findings also reveal that different strains of the same species can differ in their response to the methods used. In the second step, we compared the method developed for A. fumigatus with the method developed for Geosmithia (this study). The results (Figs.…”

Section: Resultsmentioning

confidence: 99%

“… FCM studies in fungi often use Tris buffer or Tris buffer with a chromatin stabilizer or/and chelator agent . In this study, we used TE buffer following .…”

Section: Methodsmentioning

confidence: 99%

“…The strains were treated in the same way as Geosmithia species. In the case of A. fumigatus CEA10, our method was compared with a method developed for Aspergillus fumigatus .…”

Section: Methodsmentioning

confidence: 99%

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Application of flow cytometry for genome size determination in Geosmithia fungi: A comparison of methods

et al. 2014

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Genome size has played an important role in the evolution of plants and animals because changes in genome size seem to accompany if not facilitate evolutionary adaptation to environmental conditions. Flow cytometry (FCM) is a widespread method for determining genome size thanks to its high accuracy and speed of measurements. Nevertheless, only a few comparative studies of FCM methods exist in the field of mycology, and reviews are absent. In this study, we compared the suitability of several concentrations and RNAse A incubation times, fixatives and buffers for estimating genome size in fungi. We chose the genus Geosmithia as a model filamentous fungus. We also introduced a new standard, Aspergillus fumigatus CEA10, to determine absolute genome size. We found FCM to be an appropriate method for measuring genome size in fungi, but optimization steps showed that incorrect propidium iodide staining of nuclei can overestimate genome size due to cytoplasmic staining. We identified fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl 2 buffer as the best treatment. V C 2014 International Society for Advancement of Cytometry

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“… FCM studies in fungi often use Tris buffer or Tris buffer with a chromatin stabilizer or/and chelator agent . In this study, we used TE buffer following .…”

Section: Methodsmentioning

confidence: 99%

“…The strains were treated in the same way as Geosmithia species. In the case of A. fumigatus CEA10, our method was compared with a method developed for Aspergillus fumigatus .…”

Section: Methodsmentioning

confidence: 99%

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Application of flow cytometry for genome size determination in Geosmithia fungi: A comparison of methods

et al. 2014

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“…After 5 days of cultivation at 309 C, conidia were collected with 0.85z NaCl containing 0.08z Tween 80 (Tween saline). DNA in conidia was stained with ‰uorescent dye by the method described by De Lucas et al 8) Brie‰y, conidia wereˆxed with 70z ethanol, washed with Tween saline, incubated with 1 mg W ml RNase A, and stained with 25 mg W ml propidium iodide (PI). The treated conidia were put into a ‰ow cytometer (FACScan, Becton Dickinson).…”

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confidence: 99%