Immunofluorescence and DAPI/Hoechst -fluorescence images were captured using Zeiss LSM 780 laser scanning confocal microscope (Carl Zeiss) with Plan-Apochromat 40×/1.4 DIC or 20×/0.8 DIC objective and Zeiss Zen 2011 Black software (RRID:SCR_018163). Mitotic aberrations, migrated and invaded cells, KRT14-positive and KRT14-negative cells as well as part of γH2AX foci were counted utilizing Fiji ImageJ software (RRID:SCR_002285). Most DNA repair foci were quantified using the automated AKLIDES® cell damage system (Medipan GmbH) at Universitätklinikum Hamburg-Eppendorf (Germany). Bright-field images of non-treated and DMSO/LY2109761-treated spheroids were captured using Zeiss Cell Observer spinning disc confocal microscope (Carl Zeiss) with Plan NeoFluar 10×/0.30 Ph1 objective and Zen 2012 Blue software (RRID:SCR_013672). Bright-field images of monolayer cells and esiRNA-treated spheroids were captured with VisiCam TC 20 mounted into Nicon Eclipse TE200 microscope. Spheroid images were analyzed with ImageJ.
Cell observer spinning disc confocal microscope
Manufactured by Zeiss
Sourced in Germany
The Cell Observer Spinning Disc confocal microscope by Zeiss is a high-performance imaging system designed for live-cell microscopy. It utilizes a spinning disc confocal technology to enable fast and efficient optical sectioning of samples, allowing for real-time observation of dynamic cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Zen 2011 black software, by Zeiss (1 mentions)
Zen 2012 blue software, by Zeiss (1 mentions)
Lsm 780 laser scanning confocal microscope, by Zeiss (1 mentions)
Ab5392, by Abcam (1 mentions)
Ab5905, by Merck Group (1 mentions)
A11041, by Thermo Fisher Scientific (1 mentions)
Zen black software, by Zeiss (1 mentions)
A21450, by Thermo Fisher Scientific (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Fgf basic fgf2, by R&D Systems (1 mentions)
Spheroid microplate, by Corning (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
B 27 supplement without vitamin a, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
μ dish, by Ibidi (1 mentions)
Gelatin, by Merck Group (1 mentions)
9 protocols using cell observer spinning disc confocal microscope
1
Quantitative Imaging of DNA Repair Foci
2
Autaptic Hippocampal Neuron Immunostaining
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Autaptic hippocampal neurons were fixed at DIV14 in 2% paraformaldehyde (PFA) in culture medium for 10 min and subsequently in 4% PFA for 10 min. Cells were then washed with phosphate-buffered saline (PBS), permeabilized by 0.5% Triton X-100 for 5 min, and blocked with 4% normal goat serum in 0.1% Triton X-100 (blocking solution) for 30 min. Cells were incubated with primary antibodies diluted in blocking solution (anti-MAP2, 1:500, chicken, ab5392, Abcam; and anti-vGlut1 1:1000, guinea pig, AB5905, Merck Millipore) for 2 hr at room temperature (RT). After washing with PBS, the cells incubated with secondary antibodies in blocking solution for 1 hr at RT in the dark (anti-chicken Alexa 568, 1:1000, A11041, Thermo Fisher Scientific; and anti-guinea pig Alexa 647, 1:1000, A-21450, Thermo Fisher Scientific) and washed again. Coverslips were mounted with FluorSave and imaged on Zeiss CellObserver spinning disc confocal microscope (×40 water immersion objective; NA 1.2) with Zeiss Zen Blue 2012 software. Images were acquired as Z-stack and 9 images per plane to capture the whole island in the field of view. The images were post-processed with Zeiss Zen Black software and neuronal morphology was analyzed using SynD automated image analysis (Schmitz et al., 2011 (link)).
3
3D Culture and Imaging of Cell Spheroids
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Eighteen thousand cells were plated on 200 μL of growth factor‐reduced extracellular matrix hydrogel (Cultrex, Reduced growth factor Type 2, Amsbio) in 35 mm glass‐bottom μ‐dish (Ibidi). Culturing media was MCF10A media with exceptions of 2% horse serum, 5 ng/μL EGF and 2.5% Cultrex.19 Cells were cultured for 7 days in triplicate plates. Imaging was performed with Zeiss Cell observer Spinning Disc Confocal Microscope (Carl Zeiss) using Plan NeoFluar 10×/0.30 Ph objective. The area (μm2) of 300 spheroids of both mutants and controls was analyzed with ImageJ. Lumen structure was studied by staining the spheroids with 5 μg/mL Hoechst 33342 and imaged with Plan‐Apochromat 20×/0.8 objective.
4
Oleic Acid Lipid Accumulation Assay
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HuH-7 sgRNA-ctrl or sgRNA-24 cells plated on glass coverslips and treated with Oleic Acid (diluted 1:10 in media) 24 hrs after plating. 6 hrs following Oleic Acid addition, cells were fixed using 4% PFA for 30 min at room temperature, followed by a PBS wash, and LipidTOX green neutral lipid staining (Thermo) (1:200 dilution in PBS) for 1 hr. Coverslips were mounted on glass slides using Prolong anti-fade mounting solution with DAPI. Imaging was performed on a Zeiss Cell Observer Spinning Disc Confocal Microscope and quantification was performed using ImageJ.
5
3D Meningioma Organoid Modeling
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CRISPRi-modified and fluorescently-labeled M10GdCas9-KRAB meningioma cells for 3D organoid experiments were generated by mixing sgNTC-mScarlet with sgCDKN2A-FumGW cells, sgNTC-GFP with sgARID1A-mCherry cells, or sgCDKN2A-FumGW with sgARID1A-mCherry cells 1:1. For pharmacologic experiments, a minimum of 2000 cells were seeded into each well of a PrimeSurface ultra-low attachment V-shaped 96 well plate (S-Bio, cat# MS-9096V). The following day, meningioma organoids were transferred to a spheroid microplate (Corning, cat# 4515) prior to beginning 12 days of continuous drug treatment. Organoids were maintained in a medium comprised of Advanced DMEM/F12 (Gibco, cat# 12634) supplemented with B-27 supplement without vitamin A (Gibco, cat# 12587010), N-2 supplement (Gibco, cat# 17502048), 100U/ml Anti-anti (Gibco, cat# 15240), 1% CTSTMGlutaMAXTM-1 (Gibco, cat# A1286001), 20ng/ml EGF (R&D Systems, cat# 236EG200), and 20ng/ml FGF basic/FGF2 (R&D Systems, cat# PRD23350). A Zeiss Cell Observer Spinning Disc Confocal microscope fitted with a temperature and carbon dioxide-controlled chamber was used to acquire fluorescence images of live meningioma organoids during drug treatments using Plan-Apochromat 10x/1.3 air objective.
6
Live-cell Imaging of Chromosome Segregation
7
Microglial Dynamics in Neuron Co-culture
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AH016-3 Lenti-IP-RFP-microglia were co-cultured with healthy control motor neurons in PEI- and Geltrex™-coated glass bottom dishes for confocal microscopy (VWR). The RFP signal was used to identify microglia in co-culture. To visualize microglial movement, images of the RFP signal and brightfield were taken every ~ 30 s for 1 h (2 × 2 stitched images, 20 × magnification) using a Cell Observer spinning disc confocal microscope (Zeiss) equipped with an incubation system (37 °C, 5% CO2). To image phagocytic activity, co-cultures were rinsed with Live Cell Imaging Solution (1X, ThermoFisher), and pHrodo™ Green Zymosan Bioparticles™ Conjugates (P35365, ThermoFisher) diluted in Live Cell Imaging Solution (50 µg/mL), which become fluorescent upon phagocytic uptake, were added. The dish was immediately transferred to the spinning disc confocal microscope, and stitched images (3 × 3, 20 × magnification) were acquired every 5 min for 2 h.
8
Endogenous PLK1 Knockdown Dynamics
9
Scratch Wound Healing Assay with SKOV3 and OAW42 Cells
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SKOV3 (4 × 104 cells/well) and OAW42 (3 × 104 cells/well) cells were seeded into 96-well plates, at ten replicates per sample. At about 90% confluency, a sterile 10 μL pipette tip was used to make a scratch across each well. The detached cells were removed by washing twice with a culture medium. The scratch closure was monitored during 48 h for SKOV3 and 72 h for OAW42, using a live-cell microscopy Cell Observer spinning disc confocal microscope with 10× objective magnification (100× total magnification) (Carl Zeiss, Oberkochen, Germany). Images were captured every 10 min. Image analysis was carried out with the TScratch software (CSE-lab, Zurich, Switzerland) using the default parameter settings. The experiment was repeated three times.
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