A 3DLTS was made by mixing 10,000 cancer cells with 0.5 μl Matrigel (Corning, Bedford, MA, USA). The mixture was dropped into a spherical mold and incubated at 37°C for 30 minutes to allow gelation. The mixture was then harvested in fresh medium, and type I collagen solution (5 μl, Corning) was added to fully cover the Matrigel sphere containing cells. This mixture was then dropped into a spherical mold at 37°C for 1 hour to allow gelation of the type I collagen. The 3D double-layered tumor spheroid (3DLTS) was then harvested. A single 3DLTS was cultured as a suspension in a well, and time-lapse images were collected every day for 1 week using a Nikon TE2000 microscope (Nikon) equipped with a Cascade 1K CCD camera (Roper Scientific, Tucson, AZ, USA). To assess changes in cell numbers, the PrestoBlue assay (Thermo Fisher Scientific) was applied to the 3DLTS every 3 days. To quantify cell invasion, the 3DLTS was fixed with 4% paraformaldehyde and incubated with Hoechst33342 (1:2000, room temperature for 10 min; Thermo Fisher Scientific) and then processed for microscopic examination. Fluorescence images of the stained 3DLTS were acquired using a Nikon TE2000 microscope. Stained nuclei of cells from the inner sphere that had invaded were counted using ImageJ. Each sample was assayed in three or more replicates.
Te2000 microscope
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Canada
The Nikon TE2000 microscope is a high-performance laboratory equipment designed for advanced microscopy applications. It features a modular and ergonomic design, allowing for versatile configurations to suit various research and analysis needs. The TE2000 provides precise optical performance, enabling users to capture detailed images and data with clarity and accuracy.
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Lab products found in correlation
Cascade 1k ccd camera, by Teledyne (6 mentions)
Csu x1 confocal unit, by Yokogawa (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Ixon3 electron multiplier charge coupled device camera, by Oxford Instruments (4 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (4 mentions)
Matrigel, by Corning (3 mentions)
Plan fluor oil immersion objective lens, by Nikon (3 mentions)
Orca er ccd camera, by Hamamatsu Photonics (3 mentions)
Plan apochromatic, by Nikon (3 mentions)
Primary monoclonal antibody against ki 67, by Cell Signaling Technology (3 mentions)
Nis elements software, by Nikon (3 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (3 mentions)
Ti2 microscope, by Nikon (3 mentions)
Vectashield with dapi, by Vector Laboratories (3 mentions)
Collagen type 1 solution, by Corning (2 mentions)
Prestoblue assay, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
199 protocols using te2000 microscope
1
3D Double-Layered Tumor Spheroid Model
2
3D Double-Layered Tumor Spheroid Model
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A 3DLTS was made by mixing 10,000 cancer cells with 0.5 μl Matrigel (Corning, Bedford, MA, USA). The mixture was dropped into a spherical mold and incubated at 37°C for 30 minutes to allow gelation. The mixture was then harvested in fresh medium, and type I collagen solution (5 μl, Corning) was added to fully cover the Matrigel sphere containing cells. This mixture was then dropped into a spherical mold at 37°C for 1 hour to allow gelation of the type I collagen. The 3D double-layered tumor spheroid (3DLTS) was then harvested. A single 3DLTS was cultured as a suspension in a well, and time-lapse images were collected every day for 1 week using a Nikon TE2000 microscope (Nikon) equipped with a Cascade 1K CCD camera (Roper Scientific, Tucson, AZ, USA). To assess changes in cell numbers, the PrestoBlue assay (Thermo Fisher Scientific) was applied to the 3DLTS every 3 days. To quantify cell invasion, the 3DLTS was fixed with 4% paraformaldehyde and incubated with Hoechst33342 (1:2000, room temperature for 10 min; Thermo Fisher Scientific) and then processed for microscopic examination. Fluorescence images of the stained 3DLTS were acquired using a Nikon TE2000 microscope. Stained nuclei of cells from the inner sphere that had invaded were counted using ImageJ. Each sample was assayed in three or more replicates.
3
Staining and Quantification of Myeloid Cells
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Staining of MPO+ monocytes and neutrophils was performed 24 h after IM. Midjejunal, mucosal-free muscularis whole mounts were fixed in ethanol (Applichem, Darmstadt, Germany) for 10 min and stained with Hanker-Yates reagent as described previously (24 (link)). MPO+ cells were counted under a microscope (TE2000, Nikon, Düsseldorf, Germany) in 5 randomly chosen areas in each specimen at a 200x magnification and calculated as MPO+ cells/mm2.
For immunofluorescence stainings for CX3CR1, MHCII, βIII-tubulin and IBA-1 midjejunal, mucosal-free muscularis whole mounts of either untreated CX3CR1GFP/+ or irradiated/shielded or irradiated/non-shielded CX3CR1GFP/+ were fixed in 4% PFA for 20 min, permeabilized with 1% Triton X-1000 (Sigma), blocked with 5% donkey serum for 1 h and incubated with the appropriate antibodies at 4°C overnight. For antibodies used in this study seeTable 1
For immunofluorescence stainings for CX3CR1, MHCII, βIII-tubulin and IBA-1 midjejunal, mucosal-free muscularis whole mounts of either untreated CX3CR1GFP/+ or irradiated/shielded or irradiated/non-shielded CX3CR1GFP/+ were fixed in 4% PFA for 20 min, permeabilized with 1% Triton X-1000 (Sigma), blocked with 5% donkey serum for 1 h and incubated with the appropriate antibodies at 4°C overnight. For antibodies used in this study see
4
Immunocytochemical Profiling of Astrocytes and Neurons
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Astrocyte and neuronal cultures were fixed with 4% PFA in phosphate‐buffered saline (PBS; pH 7.4) for 15 min and nonspecific sites were blocked with 3% bovine serum albumin (BSA; Sigma‐Aldrich), 5% normal goat serum (Sigma‐Aldrich) and 0.2% Triton X‐100 diluted in PBS for 1 h, before incubation with the following antibodies: rabbit anti‐GFAP (1:1000; DAKO Cytomation), rabbit anti‐lamin‐B1 (1:1000; Abcam), rabbit anti‐p16INK4a (1:100; Proteintech), rabbit anti‐iNOS (1:100; Abcam), rabbit anti‐Spinophilin (1:500; Abcam), or mouse anti‐Synaptophysin (1:1000; Millipore) at 4°C overnight. Subsequently, the cells were thoroughly washed with PBS and incubated with secondary antibodies at room temperature (RT) for 2 h. Secondary antibodies were Alexa Fluor 546‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG (1:1000; Invitrogen), or Alexa Fluor 488‐conjugated goat anti‐rabbit IgG or goat anti‐mouse IgG (1:300; Invitrogen). Nuclei were counterstained with DAPI (Sigma‐Aldrich), and cells were observed with a TE2000 Nikon microscope.
5
Quantifying Senescent Astrocyte Nuclear Deformations
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Control and senescent astrocyte cultures were immunostained for lamin-B1, counterstained with DAPI and images were acquired with a TE2000 Nikon microscope with a 63x objective. Based on the previous classification of nuclear deformations through super-resolution fluorescence microscopy and 3D reconstruction, we quantified the relative number of nuclear deformations based on the total number of lamin-B1+ nuclei per image. A total of 3,084 cells from 9 control astrocyte cultures and 4,481 cells from 10 senescent astrocyte cultures were analyzed. We also calculated the nuclear circularity per lamin-B1+ nuclei by using the Fiji software and by employing the formula: circularity = 4π (area/perimeter 2 ). Circularity has a maximum value of 1 and diminishes as the nuclear shape becomes increasingly convoluted (Zhang et al., 2018) . A total of 1,231 and 944 lamin-B1+ nuclei, respectively, from 7 and 6, control and senescent astrocyte cultures, were analyzed. Astrocytes cultures were prepared from different litters.
6
Immunofluorescence Staining of V2r in MCF-7 Cells
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Briefly, cells were seeded on glass coverslips, and fixed with paraformaldehyde. After incubation with blocking agent, cells were incubated with a goat polyclonal anti-V2r antibody for 1 h at 37°C (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Receptor-bound antibodies were detected with a secondary rabbit polyclonal FITC-conjugated antibody (Chemicon International, Temecula, CA, USA) and nuclei were labeled with DAPI (Vector Laboratories, Peterborough, UK). Samples were examined using a TE-2000 microscope (Nikon Inc., Tokyo, Japan). MCF-7 cells were used as a positive control of V2r expression (6 (link)).
7
Quantification of Autophagosome Formation
8
Imaging Microtubule Dynamics under Nucleus
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Imaging was performed on a Nikon TE2000 microscope with a Nikon A1 confocal laser unit. During microscopy, cells were maintained in an environmental chamber in which the temperature was kept at 37°C, the CO2 level at 5%, and the relative humidity at 100%. Depending on the density of microtubules under the nucleus at the start of imaging, the entire area under the nucleus was bleached to allow for easy identification of individual microtubules. After bleaching, only newly polymerized microtubule segments were fluorescent in the bleached area. Once fluorescent microtubule segments grew back into the sub-nuclear region, dashes were bleached into the microtubules, after which a time lapse image sequence was taken to capture bending events. All imaging of microtubules was performed with a 488nm wavelength laser at a power of 0.69% and a 60x oil immersion objective, with images being taken at one second intervals. Bleaching was performed at 35% laser power for one second.
9
High-throughput Yeast Phenotypic Imaging
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Images were collected using a Nikon TE2000 microscope outfitted with a perfect focus system, a Prior automated stage, a Nikon Intensilight light source, and a QI Click 2.4-megapixel camera. Typically, 65–100 fields/well were captured in two channels configured for FITC and DAPI, respectively. Unlike the original protocol (Ohya et al, 2005 (link)), ours did not include a third channel with a filamentous-actin stain (phalloidin), as we had determined that actin-based CalMorph phenotypes contributed little to analysis of morphological variation (Richardson et al, 2013 (link)). Raw tiff images were processed using custom software to produce 696 × 520 8-bit jpeg images. These images were then analyzed using the CalMorph software package (Ohya et al, 2005 (link)).
10
Microscopic Imaging of DAB-Stained Samples
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