CFBE-wt cells were seeded at a high density and grown as a confluent monolayer on glass coverslips (Fisher Scientific) for 7 days prior to use (23 (link)), at which point the cells form tight junctions that are capable of excluding bacterial transit to the bottom of the monolayer. For coinfection assays, cells were infected with RSV constitutively expressing red fluorescent protein (RFP) diluted in MEM to an MOI of 1 for 6 h. Cells were returned to growth medium for 18 h prior to inoculation with P. aeruginosa. Bacteria were inoculated at an MOI of 25. Bacteria were allowed to attach for 2 h before unattached bacteria were removed by perfusion with MEM supplemented with 2 mM l -glutamine at 20 ml/h (23 (link)). After 2 h of biofilm development, 10 random z-stacks were collected from each chamber using a Nikon Ti-inverted microscope to obtain “pretreatment” images. MEM supplemented with 2 mM l -glutamine containing vehicle or 10 µM WLBU2 was then perfused through the chamber. After 1 h of treatment, 10 random z-stacks were collected from each chamber using a Nikon Ti-inverted microscope to obtain “posttreatment” images. Biofilm biomass was quantified using Nikon Elements software (5 (link)).
Ti inverted microscope
Manufactured by Nikon
Sourced in Japan, United States, Germany
The Ti inverted microscope is a high-performance microscope designed for advanced scientific and research applications. It features a sturdy, inverted design that provides a stable platform for observing samples. The Ti inverted microscope is equipped with a range of sophisticated optics and illumination systems that enable clear, detailed imaging of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Perfect focus system, by Nikon (8 mentions)
Nis elements software, by Nikon (4 mentions)
Lsm 710 confocal microscope, by Zeiss (4 mentions)
C2 confocal scan head, by Nikon (3 mentions)
Motorized stage, by Nikon (3 mentions)
Metamorph software, by Molecular Devices (3 mentions)
Metamorph 7, by Molecular Devices (3 mentions)
35 mm glass bottomed tissue culture dishes, by MatTek (2 mentions)
Spectrax diode based light source, by Lumencor (2 mentions)
Orca flash4.0 v2 digital cmos camera, by Hamamatsu Photonics (2 mentions)
Plan fluor objective, by Sutter Instruments (2 mentions)
Xenon lamp, by Sutter Instruments (2 mentions)
Plan apo 1.4 na phase contrast objective, by Oxford Instruments (2 mentions)
Zyla 4.2 scmos camera, by Nikon (2 mentions)
Csu w1 disk, by Nikon (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Orca ag, by Hamamatsu Photonics (2 mentions)
Quad bandpass 405 491 561 642 dichroic mirror, by IDEX Corporation (2 mentions)
Et525 50 emission filter, by Chroma Technology (2 mentions)
116 protocols using ti inverted microscope
1
CFBE Monolayers Infected with RSV and P. aeruginosa
2
Immunofluorescence Imaging of Huh-7 Cells
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One day prior to study, Huh-7 cells ± CD81 and/or CD19 were seeded into standard 24-well plates at 1.2 × 104 cells/well. The cells were then fixed (in situ) in 2% formaldehyde, blocked, and stained, as described for flow cytometry, with the inclusion of a 10-min 4′,6-diamino-2-phenylindole counterstain at the end of the procedure. The samples were imaged on a Nikon Ti inverted microscope, through a 40× extra-long working distance objective, using a C2 confocal scan head with 405- and 635-nm laser illumination (Nikon Instruments, Tokyo, Japan). Multiple Z-stacks were acquired for each sample. The data were processed for display using FIJI/ImageJ (82 (link), 83 (link)).
3
Fluorescence Imaging of Circulating Tumor Cells
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Fluorescence imaging was done on a Nikon Ti inverted microscope (Swanson Biotechnology Center Microscopy Core Facility). All imaging of CTCs was performed at 40× magnification. Images were taken on a Photometrics CoolSnap HQ camera. The diameter of each cell was found by taking the maximum Feret’s diameter of the fluorescence EpCAM signal after background subtraction using ImageJ.
4
Visualization of Lymph Node Stromal Cells
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For analysis of LN stromal cells by confocal microscopy, LNs were isolated on day 7 after transplantation and frozen in OCT (Cellpath). Sections (8 μm) were cut on a cryostat, dried, and fixed with acetone (–20°C). Primary antibodies were CD31-FITC (clone MEC13.3, BD Biosciences) and gp38-biotin (eBio8.1.1, eBioscience). Secondary antibodies were anti-FITC Alexa 488 (Life Technologies) and Streptavidin eFluor 570 (eBioscience). Sections were stained with DAPI and mounted with ProLong Diamond Antifade Mountant (Life Technologies). All images were captured on a Nikon Ti inverted microscope using a C2 confocal scan head (Nikon Instruments). Images were acquired with a 40× (Plan Apochromat N.A. 0.095 W.D. 0.21 mm) objective. Image analysis was done using the software ImageJ (NIH).
5
Visualizing Viral Binding and Entry
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To study virus binding and entry by detection of viral DNA, HFFs were infected at 4°C for 1 h to allow virus binding. The cells were washed in ice-cold phosphate-buffered saline (PBS) and then either lysed immediately for analysis by qPCR for genomes (using UL138 primers as noted below) or temperature shifted to 37°C to promote viral entry. Fifteen minutes postentry, DNA was harvested from the cells and analyzed by qPCR. To assess virus binding by direct visualization of virus particles, HFFs were first labeled with 1 μg/ml Hoechst 33342 (Tocris) for 10 min at 37°C. The cells were then washed in ice-cold PBS and infected at 4°C at a multiplicity of infection (MOI) of 5 with TB40/E-UL32-GFP HCMV for 1 h. The cells were washed twice with ice-cold PBS, and live cells were visualized directly by confocal imaging using a 60× objective on a Nikon Ti inverted microscope with a C2 confocal scan head (Nikon). Z-stacks were acquired at high zoom to create representative images, while z-stacks of multiple low-zoom fields were acquired to allow quantification. ImageJ was used to process images for display.
6
Immunofluorescence and SEM Imaging of hMSCs
7
Fluorescent Protein Imaging of Mitochondria and ER
8
Quantifying Bacterial Clustering Contrast
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Cells were grown as described above using the following inducer concentrations: 0.3 μM NaSal for CheA::mYFP/CheW; 13 μM IPTG for Tar-YFP; and 50 μM IPTG for mYFP-CheR. Cells were placed on a 1% agarose pad and covered with a coverslip. Fluorescence images were obtained at 30°C using a Nikon Ti inverted microscope equipped with a 100× Plan-Fluor objective (1.3 NA), a xenon lamp (Sutter Instruments), and a camera (Andor Technology). Images were then analyzed to extract clustering contrast values by computing the ratio of peak intensity (highest) to body intensity (mean of cell without the poles) in each cell after subtraction of the background fluorescence intensity.
9
FRAP Analysis of Filopodia Dynamics
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FRAP experiment was performed on a motorized Nikon Ti inverted microscope equipped with the Perfect Focus System (Nikon, Japan) using a Plan Apo 60x/1.4 oil immersion objective lens, 491 nm laser (iLas2 FRAP-3D; Roper Scientific, France), CSU-22 spinning disk confocal scanning head (Yokogawa Electric Corp., Japan), and an Evolve EM-CCD camera (Photometrics, USA). Transiently transfected (using Lipofectamine 2000) Hela cells expressing GFP-IRSp53 and GFP-I-BAR (IRSp53 mutant) cultures were prepared in 35 mm glass bottom Willco dishes(WillCo Wells, The Netherlands) and maintained at 37 °C in 5% CO2 humidified environment using an on-stage incubator (Chamlide, Live Cell Instruments, S. Korea). Time-lapse image acquisitions were controlled using MetaMorph software (150 ms exposure, EM-gain 300) and FRAP setting was controlled through the iLas2 control window. The bleaching was carried out on filopodia with a rectangular region using 100% laser power and imaged with 15% laser power. Five pre-bleach images at 0.5 s interval was recorded before FRAP bleaching. The FRAP recovery was recorded for another 90 frames at every 0.5 s.
10
STAT3 Decoy Oligonucleotide Impacts HUVEC Motility
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5×105 HUVECs were plated in a 10 cm dish and transfected with either the STAT3 mutant control oligonucleotide or the FAM-tagged STAT3 decoy oligonucleotide. Twenty four hours after transfection, 5×104 HUVECs were plated on top of Matrigel in a 0.17 mm clear Delta T Dish (BioptechsInc, Butler, PA). The cells were placed in 37°C for 15 minutes to allow adherence and were subsequently imaged on a Nikon Ti inverted microscope with perfect focus using phase contrast at 10×. Images were captured every 10 minutes for 24 hours in 8 unique fields with a Roper Scientific Cascade 1K camera using MetaMorph 7.73 software. Cell tracking was performed with the track objects application in Metamorph to obtain mean distance traveled, mean velocity and distance from origin.
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