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Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

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Summary of the method: microencapsulation, incubation, and sorting processes. (A) A schematic representation of the flow-focusing technology is shown. The sample is injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply in a Cellena microencapsulator. The stationary jet breaks up by capillary instability into homogeneous droplets, which will gel in a continuously stirred calcium chloride solution at room temperature. Adapted from Martin-Banderas et al. (2005). (B) Spherical size-monodisperse alginate microcapsules containing the fungal spores are incubated in different media. (C) Flow cytometry and cell-sorting analysis: microcapsules containing single cell were analyzed by flow cytometry using a COPAS flow cytometer. COPAS instruments allow one to automate the process of sorting large particles (20−1500 µm) in a continuously flowing stream at a rate of 10−50 objects/sec. Via the use of object size (time of flight), optical density (extinction), and/or intensity of fluorescent markers as sorting criteria, selected objects in a predetermined range can be safely dispensed in multiwell plates for further analysis.
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fig1: Summary of the method: microencapsulation, incubation, and sorting processes. (A) A schematic representation of the flow-focusing technology is shown. The sample is injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply in a Cellena microencapsulator. The stationary jet breaks up by capillary instability into homogeneous droplets, which will gel in a continuously stirred calcium chloride solution at room temperature. Adapted from Martin-Banderas et al. (2005). (B) Spherical size-monodisperse alginate microcapsules containing the fungal spores are incubated in different media. (C) Flow cytometry and cell-sorting analysis: microcapsules containing single cell were analyzed by flow cytometry using a COPAS flow cytometer. COPAS instruments allow one to automate the process of sorting large particles (20−1500 µm) in a continuously flowing stream at a rate of 10−50 objects/sec. Via the use of object size (time of flight), optical density (extinction), and/or intensity of fluorescent markers as sorting criteria, selected objects in a predetermined range can be safely dispensed in multiwell plates for further analysis.

Mentions: Fungal spores were microencapsulated in calcium alginate beads (400 µm) in a Cellena microencapsulator device (Ingeniatrics) following the manufacturer’s instructions as previously described (Gomez-Herreros et al. 2012) (Figure 1A). Microencapsulation conditions were adjusted to obtain single-spore capsules, which ensures that all the mycelial cells growing inside the microcapsules derived from one single spore and consequently, they are clonal. This procedure gave microcapsules with a regular spherical shape and homogenous size, which contained one spore inside the beads (Figure 2A). Spore suspensions were adjusted at 0,160 units at O.D. 600 nm, and 10 µL of each suspension was mixed with 290 µL of water and 2.7 mL of alginate 3%. The sample was then injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply (Figure 1A). The stationary jet broke up by capillary instability into homogeneous droplets, which gel in a continuously stirred 3% calcium chloride solution at room temperature. The capsules were stored in the same solution at 4° with agitation for at least 1 hr or until they were used, then they were filtered and washed with distilled water to remove excess of calcium chloride. Then, microcapsules were inoculated with a sterile spoon into liquid media and incubated under the appropriate conditions.


Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Summary of the method: microencapsulation, incubation, and sorting processes. (A) A schematic representation of the flow-focusing technology is shown. The sample is injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply in a Cellena microencapsulator. The stationary jet breaks up by capillary instability into homogeneous droplets, which will gel in a continuously stirred calcium chloride solution at room temperature. Adapted from Martin-Banderas et al. (2005). (B) Spherical size-monodisperse alginate microcapsules containing the fungal spores are incubated in different media. (C) Flow cytometry and cell-sorting analysis: microcapsules containing single cell were analyzed by flow cytometry using a COPAS flow cytometer. COPAS instruments allow one to automate the process of sorting large particles (20−1500 µm) in a continuously flowing stream at a rate of 10−50 objects/sec. Via the use of object size (time of flight), optical density (extinction), and/or intensity of fluorescent markers as sorting criteria, selected objects in a predetermined range can be safely dispensed in multiwell plates for further analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4232552&req=5

fig1: Summary of the method: microencapsulation, incubation, and sorting processes. (A) A schematic representation of the flow-focusing technology is shown. The sample is injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply in a Cellena microencapsulator. The stationary jet breaks up by capillary instability into homogeneous droplets, which will gel in a continuously stirred calcium chloride solution at room temperature. Adapted from Martin-Banderas et al. (2005). (B) Spherical size-monodisperse alginate microcapsules containing the fungal spores are incubated in different media. (C) Flow cytometry and cell-sorting analysis: microcapsules containing single cell were analyzed by flow cytometry using a COPAS flow cytometer. COPAS instruments allow one to automate the process of sorting large particles (20−1500 µm) in a continuously flowing stream at a rate of 10−50 objects/sec. Via the use of object size (time of flight), optical density (extinction), and/or intensity of fluorescent markers as sorting criteria, selected objects in a predetermined range can be safely dispensed in multiwell plates for further analysis.
Mentions: Fungal spores were microencapsulated in calcium alginate beads (400 µm) in a Cellena microencapsulator device (Ingeniatrics) following the manufacturer’s instructions as previously described (Gomez-Herreros et al. 2012) (Figure 1A). Microencapsulation conditions were adjusted to obtain single-spore capsules, which ensures that all the mycelial cells growing inside the microcapsules derived from one single spore and consequently, they are clonal. This procedure gave microcapsules with a regular spherical shape and homogenous size, which contained one spore inside the beads (Figure 2A). Spore suspensions were adjusted at 0,160 units at O.D. 600 nm, and 10 µL of each suspension was mixed with 290 µL of water and 2.7 mL of alginate 3%. The sample was then injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply (Figure 1A). The stationary jet broke up by capillary instability into homogeneous droplets, which gel in a continuously stirred 3% calcium chloride solution at room temperature. The capsules were stored in the same solution at 4° with agitation for at least 1 hr or until they were used, then they were filtered and washed with distilled water to remove excess of calcium chloride. Then, microcapsules were inoculated with a sterile spoon into liquid media and incubated under the appropriate conditions.

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

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Related in: MedlinePlus