SH-SY5Y cells were fixed and stained with crystal violet (Sigma-Aldrich), phalloidin conjugated to Alexa-Fluor 488 (Invitrogen), or unstained and imaged under relief contrast conditions on an Olympus CKX41 microscope or Olympus IX81/Fluoview 1000 laser scanning confocal microscope. Images collected after staining with phalloidin (conjugated to Alexa-Fluor 488) and visualized on an Olympus IX81/Fluoview 1000 laser scanning confocal were consistent with neurons visualized by crystal violet staining or relief contrast captured on an Olympus CKX41 microscope. For quantification of neurite length/cell, after crystal violet staining, 10 fields per well were captured with a 4× objective, no contrast, for 6 wells per group. The fields, each 1,400 × 1,050 µm, were preselected in a 2 × 5 array centered in each well, with each field center 3 mm from the center of adjacent fields. Processes were traced and measured automatically with the Neurite Tracer39 (link) macros for ImageJ. Separate experiments were performed and samples were collected under relief contrast (10× objective, 560 × 420 µm fields) for determination of maximum process lengths and percentages of cells with processes by manual tracing (avoiding overlapping cells), assisted and measured with the NeuronJ plugin for ImageJ.
Ckx41 microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, Canada, United Kingdom, Australia
The CKX41 microscope is a compact and versatile inverted microscope designed for routine laboratory work. It features a built-in LED illumination system and a range of objectives to accommodate various sample types. The CKX41 provides clear, high-quality images for basic observation and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (13 mentions)
Sc30 camera, by Olympus (7 mentions)
Dfc 3200 camera, by Leica (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
Matrigel, by Corning (5 mentions)
Crystal violet solution, by Merck Group (4 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (4 mentions)
Cellsens software, by Olympus (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Analysis getit software, by Olympus (3 mentions)
Cella software version 2, by Olympus (3 mentions)
Altra 20 soft imaging system, by Olympus (3 mentions)
Transwell chamber, by Corning (3 mentions)
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (3 mentions)
Vector novared peroxidase substrate, by Vector Laboratories (3 mentions)
Anti human cd14 magnetic particles, by BD (3 mentions)
M csf, by R&D Systems (3 mentions)
Rankl, by R&D Systems (3 mentions)
Cellsens entry software, by Olympus (3 mentions)
Dp71 u tvix 2 camera, by Olympus (3 mentions)
390 protocols using ckx41 microscope
1
Neurite Quantification in SH-SY5Y Cells
2
CBD Dose-Dependent Effects on MDA-MB-231 Cells
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MDA-MB-231 cells were seeded in 60 mm plates at a density of 2 × 105 cells/mL. The day after, cells were treated with different concentrations of CBD (5, 10, 20 µM) in DMEM medium supplemented with 0.5% FBS. Then 72 h later, detached cells (derived from 10 and 20 µM CBD treatment) were collected, centrifuged (800 rpm for 5 min), resuspended in DMEM medium with 10% FBS without CBD, and plated in 60 mm tissue culture (TC) dishes. Cells were observed with an inverted Olympus CKX41 microscope (Olympus Instruments, Tokyo, Japan). Instead, cells treated with 5 µM CBD were detached with trypsin (0.25%), collected, centrifuged (800 rpm for 5 min), resuspended in DMEM medium with 10% FBS without CBD, and plated at the density of 1.6 × 105 cells/mL in 100 mm no-treated dishes. Cells were observed with an inverted Olympus CKX41 microscope (Olympus Instruments, Tokyo, Japan) and after 72 h of culture were detached with trypsin and counted. The experiments were carried out in triplicate and repeated for three independent measurements. Data were analyzed using GraphPad v6.0 software (San Diego, CA, USA) employing Student’s t-test. p < 0.05 was considered statistically significant.
3
In Vitro Wound Healing Assay for Cell Migration
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The in vitro wound healing assay for probing collective cell migration in two dimensions was performed using 2-well silicone inserts (Ibidi GmbH, Planegg, Germany) placed into a 6-well plate, which allowed the experimental variables to be standardized. To detect migration, 5 × 104 cells/well were suspended in a volume of 70 μL 10% FCS/DMEM. The cell culture inserts were removed after 24 h, leaving a defined cell-free gap of 500 µm. At this time point (0 h), the fresh medium was supplemented with medium enriched with culture fluid after a 3-day incubation with A-PRF+ alone, A-PRF+ and fibroblasts, fibroblasts alone, and DMEM alone, and then placed into each well, and images were taken.
Cell cultures were observed and photographed under the CKX41 Olympus microscope (Tokyo, Japan) after 24 and 48 h. Software ImageJ (LOCI, University of Wisconsin) was used to quantify the areas of the closing gap.
Cell cultures were observed and photographed under the CKX41 Olympus microscope (Tokyo, Japan) after 24 and 48 h. Software ImageJ (LOCI, University of Wisconsin) was used to quantify the areas of the closing gap.
4
Senescence-associated β-Galactosidase Assay
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Cells were stained for senescence-associated β-galactosidase activity using a kit from Cell Signaling Technology (#9860) as per the manufacturer’s instructions. Ten images were acquired on a CKX41 Olympus microscope 20× objective for each experimental condition. The percentages of β-galactosidase-positive cells were scored using Image J software. Results are given as means ± S.D. of four independent experiments. The in vivo senescence assay was performed in tumor sections using SenTraGor™, a Sudan Black B analog conjugated with biotin, which reacts with lipofuscin granules that have been shown to accumulate during the senescence process (39 (link)).
5
Multicellular Tumor Spheroid Cytotoxicity Assay
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For growing multicellular tumor spheroids, 400 HCT116, 400 HT29 or 800 MCF-7 cells per well were plated into round-bottom ultra-low attachment 96-well plates (Corning, Glendale, AZ, USA) in the media described above and incubated at 37 °C for 96 h. Spheroids were treated with 10 µM of the two most cytotoxic compounds 3 and 4 for another 96 h. The diameters of the spheroids were measured with Cell^F software under a CKX41 Olympus microscope before treatment and in constant daily intervals over the 96 h treatment period. For statistical analysis the t-test with Welsh’s correction was implemented in GraphPad Prism software (Version 6.01).
6
Salvianolic Acid B Enhances Wound Healing
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Based on the MTT assay results, the salvianolic acid B concentrations were selected for the wound healing assay (25 µg/mL, 50 µg/mL and 75 µg/mL). For wound healing assay fibroblasts were plated into a 6-well plate with Ibidi Culture Inserts. After 24 h, when cells had created the monolayer, the inserts were removed. The wells were filled with DMEM and salvianolic acid B in a specific concentration, then incubated for 24, 48, and 72 h. Cell culture was observed under the CKX41 Olympus microscope (Tokyo, Japan). Software ImageJ (LOCI, University of Wisconsin) was used to prepare graphics of visualization of wound closure which was created on the basis of the results of the experiments.
7
Whole-Mount Embryo Staining Protocol
8
Cell Morphology and Proliferation Assay
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Cell morphology and pigmentation were examined under a CKX41 microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Cell cultures were analyzed on day 1 by removing the medium and adding an MTT solution (0.5 mg/mL in phenol red-free culture medium). After 2 h of incubation, the solution was replaced with 200 μL DMSO. After 10 min, the absorbance was determined at 570 nm (ref. 630 nm [36 (link)]) by a microplate spectrophotometer (Dai-Nippon Pharmaceutical Co., Osaka, Japan). The cell proliferation of each group was calculated as the absorbance of the treatment group relative to the control.
9
Histological Analysis of Liver Tissue
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Liver tissues were removed from a portion of the left lobe, fixed immediately in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5-μm thickness. Serial sections were stained with hematoxylin and eosin for evaluation of portal inflammation, hepatocellular necrosis, and inflammatory cell infiltration. Sections were examined in a blinded manner under an Olympus CKX 41 microscope (Olympus Optical Co. Ltd., Tokyo, Japan).
10
Quantifying LEC Lumen Area Dynamics
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Images for quantifying LEC lumen area were taken on an Olympus CKX41 microscope (Olympus) and imaging software (Olympus). Time-lapse movies of LECs were taken using a DMI6000B microscope with environmental chamber (Leica Microsystems) and controlled using MetaMorph 7.8 software (Molecular Devices). A ×10 objective was used for all movies. Time lapse movies were created by taking an image every 10 min with a monochromatic Hamamatsu ORCA-ER C4742-80 camera (Hamamatsu) over 0–48 and 48–72 h. Using MetaMorph software, these images were compiled into a movie. Tube areas were also evaluated using MetaMorph. Movies were processed, edited, and stabilized using Adobe Creative Cloud: Adobe After Effects (Adobe Systems). All confocal images were taken on a Leica SP8 LIGHTNING White light laser confocal scanning microscope. Confocal images were taken using either a 10X HC PL APO, 0.4NA (numerical aperture), WD (working distance) 2.2 mm or 40X HC PL APO, water immersion, 1.1NA,WD 0.65 mm lenses. Confocal z-stack reconstructions were created by either using LAS X (Leica Microsystems) or Fiji [(Fiji is just) ImageJ].
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