Pore cell strainer

B. cinerea conidia were harvested in 1 mg mL⁻¹ glucose and 1 mg mL⁻¹ K₂HPO₄ and filtered through a 40-μm pore cell strainer (Corning). Spore concentration was adjusted to 10⁶ spores mL⁻¹ using a Neubauer chamber. B. cinerea spores were grown in liquid PDB, at full strength for 6 h, or 1/4 strength for 24 h. 10 μM iP-NBD was then added to the germlings, which were grown with the fluorescent CK for an additional 2 h. The fungal matter was then washed in water, resuspended in 1 mg mL⁻¹ glucose and 1 mg mL⁻¹ K₂HPO₄, and mounted for confocal microscopy as detailed above, using the GFP track. Internalized CK was quantified from 17 images per treatment, using Fiji-ImageJ. Mean fluorescence was calculated using the measure tool, and the corrected total cell fluorescence was calculated as the Integrated Density – (Area of selected cell X Mean fluorescence of background readings).

To evaluate the effect of CK on glucose utilization, the decline in glucose levels in the medium following fungal growth was measured. B. cinerea conidia were harvested in 1 mg mL⁻¹ glucose and 1 mg mL⁻¹ K₂HPO₄ and filtered through a 40-μm pore cell strainer (Corning). Spore concentration was adjusted to 10⁶ spores mL⁻¹ using a Neubauer chamber. B. cinerea spores were grown in liquid media: PDB or defined media as detailed in Table 1 above. A total of 100 μM CK was added to both PDB and defined media cultures, which were then allowed to grow for 48 h. The amount of metabolized glucose was analyzed by measuring the amount of glucose present in the media by the 3, 5 Dinitrosalicylic acid (DNSA) method (66 (link)), using dextrose as a standard. 3,5-Dinitrosalicylic acid was purchased from Sigma-Aldrich. The utilized glucose was assumed to be inversely proportional to the amount present in the medium. For control, glucose was measured in the different media, after they were subjected to the above-mentioned conditions, without B. cinerea.

To evaluate the effect of CK on glucose utilization, the decline in glucose levels in the medium following fungal growth was measured. B. cinerea conidia were harvested in 1 mg mL⁻¹ glucose and 1 mg mL⁻¹ K₂HPO₄ and filtered through a 40-μm pore cell strainer (Corning). Spore concentration was adjusted to 10⁶ spores mL⁻¹ using a Neubauer chamber. B. cinerea spores were grown in liquid media: PDB or defined media as detailed in Table 1 above. A total of 100 μM CK was added to both PDB and defined media cultures, which were then allowed to grow for 48 h. The amount of metabolized glucose was analyzed by measuring the amount of glucose present in the media by the 3, 5 Dinitrosalicylic acid (DNSA) method (66 (link)), using dextrose as a standard. 3,5-Dinitrosalicylic acid was purchased from Sigma-Aldrich. The utilized glucose was assumed to be inversely proportional to the amount present in the medium. For control, glucose was measured in the different media, after they were subjected to the above-mentioned conditions, without B. cinerea.