Hcx plan apochromat 100

After incubation in the appropriate medium, ∼1.5 OD_600nm of cells were harvested by centrifugation at 3,000 g for 3 min, resuspended in a small volume of the corresponding medium, spotted on a microscopy slide, and subsequently live-imaged at 25°C with a DMI6000 B epifluorescence microscope (Leica) equipped with a DFC 360FX camera (Leica) using an HCX Plan Apochromat 100× 1.4 NA objective lens. Images were acquired using LAS AF software (Leica) with 0.5–2 s exposure times. For the quantification of the localization phenotypes of Grh1-2×GFP, whole cell z stacks with a step size of 0.4 µm were captured as described above. The corresponding maximum-projection images were created with ImageJ 1.45r and the number of cells showing the following phenotypes for the distribution of Grh1-2×GFP was manually counted: diffuse, growth (>5 small punctate structures), CUPS (1–3 large dots), or intermediate (1–3 large dots and several small punctate structures). At least three microscopic fields (≥50 cells) were examined for each condition. Results represent the percentage of cells with each distribution phenotype and are representative of at least two experiments.