Five days after shRNA transduction, cells were fixed with 4% PFA and stained with the Senescence β‐Galactosidase Staining Kit (Cell Signaling). Images of 10 random fields per sample were taken with a Zeiss Cell Observer microscope at ×100 magnification. The staining intensity of senescent cells was determined using Fiji software (Schindelin et al, 2012 ). Normalized staining intensities were calculated dividing the total intensity by the number of β‐galactosidase‐positive cells.
Cell observer microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss Cell Observer is a high-performance microscope system designed for live-cell imaging and analysis. It features advanced optical components and digital imaging capabilities to provide clear, detailed images of cellular structures and processes.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (6 mentions)
Axiocam hrm camera, by Zeiss (4 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Ab15277, by Abcam (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Bodipy fl, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Zeiss (1 mentions)
Matrigel, by Corning (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti paxillin, by BD (1 mentions)
Ln121, by BioLamina (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
56 protocols using cell observer microscope
1
Senescence β-Galactosidase Staining
2
Live-cell Microscopy of siRNA Transfection
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siRNA-transfected cells were cultured in a 37 °C microscope chamber with 5% CO2. Cells were viewed with a Zeiss Cell Observer microscope. The time-lapse series was acquired on a Cell Observer system consisting of a fully motorized Axiovert 200M microscope, ×20 objective, Axiocam HRm and AxioVision 3.1 using the Time-lapse modules. Imaging was performed for 24 h with a lapse time of 8 min.
3
Time-lapse Analysis of Cell Cycle Dynamics
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Cell cycle parameters, cell area, mode of cell division and cell survival were analyzed by time-lapse video microscopy (Costa et al., 2008 (link), 2011 (link)). Briefly, cultures were imaged every 4 min using a Cell Observer microscope (Zeiss) and a self-written VBA module remote controlling Zeiss AxioVision software (Rieger et al., 2009 (link)). Images were assembled into a movie using the software Timm's Tracking Tool—TTT (Rieger et al., 2009 (link)), allowing the identification and tracking of individual clones. Mode of cell division was classified based on the behavior of daughter cells in: Symmetric Progenitor (both daughter cells continue to proliferate), Asymmetric (one daughter cell continues to proliferate and the other becomes post-mitotic), or Symmetric Terminal (both daughter cells become post-mitotic). Cell cycle length was measured as the time spanned by proliferating cells between their generation and division. Cell size was measured as the area covered by the cell's soma (in μ m2) 10 min prior to division. Cell survival was quantified every 12 h for each cell lineage. Briefly, the number of cells alive at 12, 24, 36, 48, 60, 72, and 84 h was divided by the total number of cells generated before these time-points within individual clones.
4
Subconfluent shAsc-1 Cell Imaging
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Subconfluent shAsc-1 cells were cultured in regular growth medium supplemented with 170 nM insulin. Imaging was initiated 4 h post seeding and continued for 3 days. The live cell imaging was performed with the Zeiss Cell Observer microscope using the Zen 2.6 program. Pictures were taken in 10 min intervals.
5
Microscopic Analysis of Blood Samples
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In preparation for microscopy, samples were gently mixed. When the blood pellet was fully re-suspended, 5 μL of the solution was applied to a glass slide and examined under a #1.5 coverslip after settling for a few minutes.
All microscopy was performed at the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. Samples were examined as a wet-preparation using an oil-immersion objective (×100/1.46) in an inverted Zeiss CellObserver microscope with a DIC setup (condenser NA = 0.55). DIC images of all samples were recorded using a Hamamatsu Orca LT camera.
All microscopy was performed at the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. Samples were examined as a wet-preparation using an oil-immersion objective (×100/1.46) in an inverted Zeiss CellObserver microscope with a DIC setup (condenser NA = 0.55). DIC images of all samples were recorded using a Hamamatsu Orca LT camera.
6
Quantitative Analysis of LDL Uptake
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LDL uptake was measured using using fluorescently labelled Low Density Lipoprotein from Human Plasma, BODIPY® FL complex (Thermo Fisher Scientific # L3483) according to manufacturer’s protocol. In brief, HUVECs were reverse transfected with indicated siRNA oligos and seeded at 1 × 104 on gelatin coated chamber slides. Cells were changed to lipid-deprived media containing 2% lipid depleted serum 24 h before harvest. Cells were labelled with 15 µg/mL BODIPY® FL for 30 min on ice and chased for 10 min at 37 °C. Cells were fixed in 4% paraformaldehyde on ice, washed three times in PBS and counterstained with Hoechst in PBS. The intensity was quantified from an average of 500 cells per experiment and each experiment represent cells pooled from four individual donors. For quantitative analysis, cells were imaged on a Zeiss Cell Observer microscope. The Zen Blue software (Zeiss) was used for automated capture of 40 images per sample with a 20x magnification. A pipeline was created in Cellprofiler39 (link),40 (link) to count dots and to measure intensity per cell. KNIME was used for data mining and analysis41 .
7
Matrigel-Based Tube Formation Assay
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The neovascularization assays were performed in 24-well plates coated with Matrigel (Corning, NY). hCMEC/D3 cells (3 × 105) were plated on Matrigel and incubated for 18 h. Tube formation was observed at 5X using an inverted fluorescence Cell Observer microscope (Carl Zeiss, Jena, Germany). Length of tubes was measured in four independent fields using the “Angiogenesis Analyzer” software from Image J (National Institutes of Health).
8
Integrin-Mediated Cellular Adhesion and Apoptosis
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Either LN111 or LN121 (both 10 μg ml–1, Biolamina) was loaded on coverglass slips precoated with PLL (Sigma), and SUM159-LM1 cells were then seeded onto the coverslips. To inhibit integrin β1 function, anti-integrin β1 blocking antibody (2.5 μg ml–1, Merck) was used. After 24–48 h, cells were fixed with 2% formaldehyde/PBS on ice, permeabilized with 0.1% Triton X-100 and 0.1% Tween20, blocked and incubated with either anti-paxillin (1:1,000, BD Transduction Laboratories) or anti-cleaved caspase 3 (1:250, Cell signaling). Cy3- or Alexa-488-conjugated secondary antibodies (1:500, Invitrogen) were used to reveal staining and DAPI (BioLegend) to stain nuclei. Cells were imaged using a Cell observer microscope (Zeiss), and cell area and number of apoptotic cells per field were analyzed with FIJI (ImageJ).
9
Viral Entry Kinetics Visualization
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To synchronise viral entry events, SVG cells were spinoculated at 4°C for 1 h with the EGFP content-labelled HIV-1 YU2ciGFP, followed by extensive washing and fixation with 4% paraformaldehyde (1 × PBS) at 0, 15, 45 and 135 mins post-infection. Samples were immunofluorescently stained for vesicle and endosomal markers using mouse anti-human antibodies specific for CD81, EEA1, CD63, CD107b and isotype control (JS-81, 14/EEA1, H5C6, H4B4, MOPC-21, clones respectively) (Becton Dickinson, NJ, USA) at 1∶200 and goat-anti mouse Alexa Fluor 555 (Life Technologies) at 1∶400. Nuclei were counterstained using Hoechst 33258 (Life Technologies). Samples were imaged on a Zeiss Cell Observer microscope using an air objective (40×, NA 0.75). IMARIS software (Bitplane, Zurich, Switzerland) was used to analyse images and quantify co-localization using the Coloc module as previously described [39] (link). Multiple fields of view and three independent experiments were used to generate the data.
10
Quantifying TRIM32 and PKCζ in CD4+ Cells
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