OVCAR8 and OVCAR8/ADR ovarian cancer cells were grown to 50–60% confluency on an eight-well chambered LabTek II coverglass, treated with appropriately labeled dye-conjugate, and incubated for 6 h. Cells were washed with PBS three times and resuspended in fresh media. Live cell imaging was performed using an inverted Zeiss LSM 700 confocal microscope equipped with a CO2 module, heating unit, and heating plate using a 40×/0.75 M27 EC Plan-Neofluar objective. Imaging was carried out at 37 °C in 5% CO2 with cells plated in a LabTek II coverglass. Images were acquired and processed with the Zeiss Zen 2009 image software. The fluorescence micrographs shown are representative of at least three independent experiments. Average fluorescence intensity was quantified using Zen 2009 software.
Zen 2009 image software
Manufactured by Zeiss
Sourced in Germany
Zen 2009 image software is a comprehensive image processing and analysis platform developed by Zeiss. It provides a suite of tools for managing, visualizing, and analyzing microscopy images. The software supports a wide range of image formats and enables users to perform basic image editing, advanced segmentation, and quantitative analysis tasks.
Automatically generated - may contain errors
3 protocols using zen 2009 image software
1
Live Cell Imaging of Ovarian Cancer Cells
2
Mitochondrial ROS Detection Assay
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Cells (1× 105/sample) were cultured at 37°C in culture medium w/o BCS in the presence of 250 nM Mitotracker Orange (Invitrogen, Molecular Probes, #M7511) in the dark, then washed with PBS and treated for 20 min in culture medium w/o BCS at 37°C in the presence of 100 μM GroPIns, 35 μM Fludarabine or the combination of both. Diagnostic microscope slides were coated with polylysine (Sigma-Aldrich, #1274) and treated cells were allowed to adhere for 10 min. Slides were immediately fixed in methanol (Carlo Erba, #412383) at -20°C for 10 min as described (33 (link)). Following fixation, samples were washed 5 min in PBS and incubated with anti-Bax (B-9) primary antibodies o/n at 4°C or 1 h at RT. After washing in PBS, samples were incubated for 1 h at RT with Alexa Fluor 488-labeled secondary antibodies. Confocal microscopy was carried out on a Zeiss LSM700 using a 63× objective, as reported (33 (link)). Images were processed with Zen 2009 image software (Carl Zeiss, Jena, Germany) and analyses were performed using ImageJ software (downloaded from http://www.embl-heidelberg.de/eamnet/ ).
3
Visualizing Intestinal Colonization by S. aureus
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To confirm the intestinal colonization, C. elegans N2, oga-1 and ogt-1 mutants were exposed to S. aureus for 72 h. After infection, worms were washed thrice with M9 buffer containing 1 mM sodium azide and stained with 0.1% acridine orange solution for 5 min. After staining, the worms were washed twice with M9 buffer to remove left-over stain. Then, the worms were placed on microscopic slides, covered with cover slips and visualized under CLSM [(LSM 710, Carl Zeiss, Germany) (10×, dry, EC Plan-Neofluar objective lens with 0.3 NA and 10× optical lens)] using 488 nm argon laser to excite the acridine orange and a 500 to 600 nm band pass emission filter for the detection of the fluorescence signal as mentioned in our previously published reports.39,44 (link) The fluorescence image acquisition was done using Zen 2009 image software (Carl Zeiss, Germany) in which the scan speed was set to 2 s, bit depth 12 and pin hole was set 1 Airy Unit (AU) as the parameters.45 (link) The gain master, digital gain and digital offset were set constant for each sample. Further, the Transmitted-Photo Multiplier Tube detector (T-PMT) mode was also enabled in the software to acquire the bright field images. The presence of acridine orange fluorescence in the pharyngeal and intestinal region indicates the colonisation of S. aureus.
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