For the wound-healing assay, cells were cultured in 24-well plates (1 × 105 cells per well) for 24 h to achieve 90% confluence. A vertical or horizontal wound was created by using a SPLScar scratcher (SPL, Korea). The wounded cells were washed once with DMEM or RPMI 1640 with 10% FBS and allowed to grow in the medium for 24–48 h. Images of cells were taken with a Leica DMi8 inverted light microscope and quantitatively analyzed using ImageJ 1.51s software (https://imagej.nih.gov/ ) installed with a macro (edge-detection/thresholding method) for automated analysis of scratch-wound assays.
Dmi8 inverted light microscope
Manufactured by Leica
Sourced in Germany
The DMi8 is Leica's inverted light microscope designed for advanced imaging applications. It features a robust and modular design, allowing for customization to meet the specific needs of research laboratories and imaging facilities. The core function of the DMi8 is to provide high-quality optical performance and versatility for a wide range of microscopy techniques, including brightfield, phase contrast, and fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Splscar scratcher, by SPL Life Sciences (1 mentions)
Spark plate reader, by Tecan (1 mentions)
Cm1800, by Leica (1 mentions)
Application suite x software, by Leica (1 mentions)
Hematoxylin, by ScyTek Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Biosesang (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
5 protocols using dmi8 inverted light microscope
1
Automated Wound Healing Assay
2
Measuring Intracellular ROS in SH-SY5Y Cells
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Intracellular ROS levels were detected using a H2DCFDA dye method. Differentiated SH-SY5Y cells were seeded in 24 well plates (2 × 104 cells/well) and 10 μM dye was added for 30 min at 37 °C in a CO2 incubator before treatment. From the DCF fluorescence, we measured intracellular ROS with a Leica DMi8 inverted light microscope with Leica Application Suite X software to process the image. The mean gray values of images were measured and quantified in >10 randomly selected images using Image J software.
3
Comparative Analyses of Digital and Analog RCP Quantification
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In order to compare digital and analog measurements of RCPs, we coated a microplate (Cell culture, 96 wells, PS half area, μ-clear, Black Advanced TC; Greiner Bio-One) with 200 nM recombinant streptavidin (Roche), diluted in coating buffer (bicarbonate/carbonate 100 mM pH 9.6) and incubated for 2 h at RT. The coating solution was then removed and the wells were washed with PBS. A dilution series between 100 pM and 100 aM of DNA circles hybridized to biotinylated primers was prepared in duplicate in Binding & Wash buffer. Immobilization, RCA and staining were performed as described above using volumes of 100 μl per well. Washes were performed with 200 μl of Binding & Wash buffer.
A Tecan Spark plate reader was used for analog readout, selecting a monochromatic acquisition (575–620 nm; band width 20 nm) and fluorescence bottom reading. Digital readout was performed on a Leica DMi8 inverted light microscope using a 20× dry objective (0.75.dry UV HC, PL APO CS2) and a filter set for TexasRed. Exposure times and imaging conditions were kept constant. Images were exported in TIFF format and analyzed with CellProfiler.
This entire experiment has been performed twice with closely similar results.
A Tecan Spark plate reader was used for analog readout, selecting a monochromatic acquisition (575–620 nm; band width 20 nm) and fluorescence bottom reading. Digital readout was performed on a Leica DMi8 inverted light microscope using a 20× dry objective (0.75.dry UV HC, PL APO CS2) and a filter set for TexasRed. Exposure times and imaging conditions were kept constant. Images were exported in TIFF format and analyzed with CellProfiler.
This entire experiment has been performed twice with closely similar results.
4
Quantitative Immunofluorescence Analysis of Spinal Cord and Neuronal Inflammation
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The L4–L6 spinal cord regions were isolated and fixed in 10% neutral buffered formalin for 2 h at 4 °C. Subsequently, the spinal cord samples were equilibrated in a 30% sucrose solution at 4 °C overnight. The tissues were embedded in optimal cutting temperature (OCT) compound and flash-frozen in liquid nitrogen. Tissue sections were sliced at a thickness of 12 µm by a cryostat (Leica CM1800; Heidelberg, Germany). SH-SY5Y cells were seeded on glass coverslips and fixed with 10% neutral buffered formalin after treatment, washed twice in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS. Samples were blocked with 3% bovine serum albumin in PBS and incubated with anti-CGRP, anti-TNF-α, anti-IL-1β and anti-phospho-NF-κB primary antibodies for 16 h at 4 °C. Next, the slides were incubated with Alexa Fluor 488 goat anti-rabbit IgG or FITC-conjugated IB4 for 1 h at room temperature. Slides were mounted with Fluoroshield with DAPI. Images were acquired by a Leica DMi8 inverted light microscope with Leica Application Suite X software (Version 3.0.3) (Leica, Wetzlar, Germany) to process the image. The mean gray values of images or phosphor-NF-κB puncta were measured and quantified in >10 randomly selected images using Image J software.
5
HeLa Cell Invasion Assay Protocol
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HeLa cells were plated on a transwell of BioCoat Matrigel invasion chambers (8.0-μm polyester [PET] membrane; Corning Life Sciences, USA) in a 24-well plate (50 × 105 cells per well) and reverse transfected using RNAiMax (Invitrogen, USA) with 50 nM RNA duplexes, according to the manufacturer’s protocol. After 24 h, the cells in the transwell were changed to grow in the media without FBS. The lower chamber contained media with 10% FBS to serve as chemoattractant. After 48 h, non-invading cells were removed with cotton swabs. Then, invading cells in Matrigel-precoated PET membranes were visualized by hematoxylin and eosin (H&E) staining: the membranes were fixed with 100% methanol (Biosesang, Korea) for 5 min after the PBS (Biosesang, Korea) wash. After a quick wash with distilled water, the membranes were stained with hematoxylin (ScyTek Laboratories, USA) for 5 min, again washed with distilled water, and stained with eosin (Sigma-Aldrich, USA) for 1 min. The membranes were mounted on a glass slide after double washing with distilled water. Then, the stained cells were examined and quantitated using a Leica DMi8 inverted light microscope.
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