Cells were grown in SCD or YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM for 30 minutes, after which the indicated amino acids were added. Cells were collected by centrifugation (600 x g, 2 min) and subjected to microscopy. The cells were observed on a Leica AF6500 fluorescence imaging system (Leica Microsystems) mounted on a DMI6000B microscope (HCX PL APO 100/1.40–0.70 oil-immersion objective lens, xenon lamp (Leica Microsystems)) under the control of the LAS-AF software (Leica Microsystems). For time-lapse imaging, the cells were grown in YMM medium containing ammonium sulfate as the sole nitrogen source, and then shifted to nitrogen-free YMM on a glass bottom dish (Matsunami Glass) mounted with 2 mg/ml of concanavalin A (Sigma). Cells were then subjected to time-lapse imaging after addition of glutamine (final concentration of 0.5 mg/ml). Images were recorded using a DeltaVision Personal system (Applied Precision) mounted on a IX71 microscope (UPlanSApo 100x/1.40 oil-immersion objective lens, LED lamp (OLYMPUS)). ImageJ software (National Institutes of Health) was used to process and produce merged images.
Uplansapo 100x 1
Manufactured by Olympus
The UPlanSApo 100x/1.40 is a high-performance objective lens designed for microscopy applications. It provides a magnification of 100x and a numerical aperture of 1.40, enabling high-resolution imaging and detailed observation of samples.
Automatically generated - may contain errors
2 protocols using uplansapo 100x 1
1
Imaging of Nitrogen Starvation Response
2
Microscopic Visualization of Exocyst Dynamics
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Microscopy was conducted with a fluorescence microscope (OLYMPUS) equipped with a UPlanSApo 100X/1.40 oil immersion objective lens. Images were taken using Image-Pro Plus 7.0 acquisition software. Determination of the cell cycle stages was performed by surveying nuclear position and morphology using a MARS-tagged nuclear envelope protein (Nup57-MARS), as described previously [22 (link)]. The Nup57-MARS yeast strains harboring either Sec5-3GFP, Exo70-3GFP, or Exo84-3GFP on the chromosome were grown in synthetic complete SD medium overnight at 25°C. Cells were collected by centrifugation and resuspended in fresh SD medium.
To examine the co-localization of exocyst subunits, cdk1-as mutant cells carrying Sec3-tdTomato and Exo84-3GFP on the chromosomal were grown to early log phase at 25°C, followed by either DMSO or 15μM 1NM-PP1 for 30 min. The cells were then fixed for fluorescence microscopy. The GFP signal was visualized with a 485/30-nm band-pass excitation filter, and a 525/25-nm band-pass emission filter. The tdTomato fluorescence was visualized with a 560/25-nm band-pass excitation filter, and a 600/50-nm band-pass emission filter.
To examine the co-localization of exocyst subunits, cdk1-as mutant cells carrying Sec3-tdTomato and Exo84-3GFP on the chromosomal were grown to early log phase at 25°C, followed by either DMSO or 15μM 1NM-PP1 for 30 min. The cells were then fixed for fluorescence microscopy. The GFP signal was visualized with a 485/30-nm band-pass excitation filter, and a 525/25-nm band-pass emission filter. The tdTomato fluorescence was visualized with a 560/25-nm band-pass excitation filter, and a 600/50-nm band-pass emission filter.
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