Exponentially growing cultures of S. cerevisiae BY4741 (OD600 of 0.8) were exposed to 500 µg·mL−1 of NME for 120 min. An aliquot was harvested, centrifuged (7100× g, 6 min), resuspended in 1× phosphate-buffered saline (PBS), and subjected to exposure to 5 µg·mL−1 PI (Invitrogen, Waltham, MA, USA) for 20 min in the dark and at room temperature. Subsequently, cells were examined by fluorescence microscopy using a Leica DM-5000B microscope (Leica Microsystems, Wetzlar, Germany). Images were captured with a Leica DCF350FX digital camera and processed using LAS AF Leica Microsystems software v. 4.0.11706. The percentage of PI-labeled cells was calculated based on the number of PI-positive cells out of a total of 200 to 300 cells per replicate under each condition.
Dcf350fx
Manufactured by Leica
The DCF350FX is a high-precision laboratory equipment designed for advanced microscopy and imaging applications. It features a robust and durable construction with a compact and ergonomic design. The core function of the DCF350FX is to provide consistent and reliable performance for various microscopy-based research and analysis tasks.
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3 protocols using dcf350fx
1
Yeast Viability Assay with PI
2
Quantifying Mitotic and Adhesion Phenotypes
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Mitotic and adhesion phenotypes were quantified by epifluorescence microscopy under a fluorescent (DM5500B; Leica) microscope using a 20×/NA 0.15 objective lens at room temperature. All antibodies used for immunofluorescence were employed according to manufacturer’s instructions (a complete list is reported in the Supplemental Experimental Procedures ). Fluorochromes used were Cy3 (cyclin B, lamin B, vinculin, pMLC2, Flag-tag), Alexa488 (pH3), FITC-/TRITC-phalloidin, or Cy5 (vinculin [pH3], Myc-tag). All images were acquired with a camera (DCF350FX; Leica) and LAS-AF image software (Leica). Images were processed with the same settings (brightness, contrast, crop, image size) using Adobe Photoshop CS5.1, and imaged figures were constructed in Adobe Illustrator.
Confocal analysis of DEPDC1B localization, actin cytoskeleton, and FAs was performed on a Leica TCS SP2 AOBS microscope, using a 40×/NA 1.25 oil-immersion objective and processed in Adobe Photoshop. Images were taken with identical settings, and the number and average area of FAs per cell were determined using ImageJ software with a mask with a fixed threshold that identifies vinculin- or paxillin-GFP positive FAs. Details on live microscopy (time-lapse and TIRF) are in theSupplemental Experimental Procedures .
Confocal analysis of DEPDC1B localization, actin cytoskeleton, and FAs was performed on a Leica TCS SP2 AOBS microscope, using a 40×/NA 1.25 oil-immersion objective and processed in Adobe Photoshop. Images were taken with identical settings, and the number and average area of FAs per cell were determined using ImageJ software with a mask with a fixed threshold that identifies vinculin- or paxillin-GFP positive FAs. Details on live microscopy (time-lapse and TIRF) are in the
3
Evaluating Apoptosis in M. avium-Infected Macrophages
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Evaluation of apoptotic cell death was performed using an in situ cell death detection kit (Roche, UK), based on the Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay. BMMΦ were seeded on top of coverslips, in a 24-well plate, at 5 × 10 5 cells/well. Infection with M. avium was performed at a multiplicity of infection (M.O.I.) = 2 and both infected and non-infected cells were treated as described in the sections above. After 24 h, the cells were fixed using 2% paraformaldehyde (PFA) and TUNEL was applied according to the manufacturer's instructions. The coverslips were observed under a fluorescence microscope (Leica DMIRE2, with a DCF350FX camera) and at least 300 cells were counted per condition.
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