The in vitro wound-healing assay was used to assess directional cell motility in two dimensions. MDA-MB-231 cells were plated in sterile 24-well plates (6 replicates/group) and allowed to form a confluent monolayer per well overnight. Wounds were then inflicted in each cell monolayer using a sterile 200 μL pipette tip. The media was removed and cells were washed twice with PBS and once with fresh RPMI medium to remove cell debris. Cells were then incubated with HVS at the desired concentrations in serum-free defined media containing 40 ng/mL HGF. Cells were incubated for 24 h and afterward, media was removed and cells were washed with pre-cooled PBS, fixed with methanol previously cooled to −20°C, and stained with Giemsa. Wound healing was visualized at 0 and 24 h by Nikon ECLIPSE TE200-U microscope and digital images were captured using Nikon NIS Elements software (Nikon Instruments Inc., Melville, NY). The distance traveled by the cells was determined by measuring the wound width at 24 h and subtracting it from the wound width at the start of treatment (t0, zero time). The values obtained were then expressed as % migration, setting the gap width at t0 as 100%. The distance migrated was calculated in three or more randomly selected fields per treatment group.
Eclipse te200 u microscope
Manufactured by Nikon
Sourced in United States
The Eclipse TE200-U is a research-grade microscope designed for a variety of laboratory applications. It features a high-quality optical system, including a sturdy frame and a range of magnification options. The microscope is capable of various imaging techniques, such as brightfield, phase contrast, and fluorescence, making it suitable for a wide range of sample types and research purposes.
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Lab products found in correlation
Nis elements software, by Nikon (4 mentions)
Eagle s minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Prolong gold mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Suramin, by Merck Group (1 mentions)
Ar c 118925xx, by Bio-Techne (1 mentions)
Brilliant blue g, by Merck Group (1 mentions)
Mrs2768, by Bio-Techne (1 mentions)
A740003, by Bio-Techne (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Cytoselect 24 well cell migration and invasion assay kit, by Cell Biolabs (1 mentions)
Versamax tunable microplate reader, by Molecular Devices (1 mentions)
12 protocols using eclipse te200 u microscope
1
In Vitro Wound Healing Assay
2
Organoid Viability Monitoring Assay
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PER2::LUC enteroids were plated in Matrigel™ with mouse Noggin and mouse EGF. R-spondin was added to the medium instead of Matrigel™ to ensure complete washout. Samples were serum shocked for 2 hours. Serum shock was then replaced with minigut medium plus R-spondin for both the control and 24-hour washout samples, or minigut medium alone for the No R-spondin samples. After 24 hours, all samples were washed with 1 ml warm PBS then refreshed with minigut medium with Noggin and EGF. R-spondin was added back only to control samples. Brightfield images (4× magnification) were taken immediately following washout, then again on Day 3 and Day 5 to monitor viability under the three conditions using the Nikon ECLIPSE TE 200-U microscope.
3
Galectin-3 Binding Assay for Cell Aggregation
4
Neurite Outgrowth Analysis in PC12 Cells
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PC12 cells plated onto collagen-coated coverslips in 12-well dishes were transiently transfected and stimulated with NGF (100 ng/ml) or EGF (100 ng/ml) for 72 h. Cells were washed with phosphate-buffered saline before and after being fixed with 4% paraformaldehyde (10 min) and permeabilized with 0.2% Triton X-100 (10 min). Transfected cells were detected by GFP-fluorescence. Alternatively, cells were incubated with anti-AU5 mAb (1 h, room temperature). After several washes, cells were incubated with Alexa 488-conjugated secondary antibodies (Molecular Probes, Invitrogen; 1 h, room temperature). Cells (50 to 100/condition) were analyzed in a Nikon Eclipse TE200-U microscope. Cells with and without neurites were then counted. Total neurite length from each cell was measured using ImageJ software (National Institutes of Health). Image capture and analyses were performed blind to the experimental conditions. Neurite outgrowth was defined as a process equal to or greater than one cell body in length for EGF treatment, and equal to or greater than two cell bodies in length for NGF treatment.
5
Immunohistochemical Analysis of Dopaminergic Neurons
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For histological analysis, the mice were intracardially perfused with 0.9% NaCl and fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Cryoprotected brains were serially sectioned at 40 μm in the coronal plane using a freezing microtome (MICROM; Walldorf, Germany). The sections were immunostained for STR and substantia nigra (SN) using anti-tyrosine hydroxylase (TH; a dopaminergic neuronal marker) as previously described [24 (link)]. Images were acquired using a Nikon ECLIPSE TE 200-U microscope (Tokyo, Japan).
6
Wound Healing Assay for MDA-MB-231 Cells
7
Assessing Tumor Cell Proliferation
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Tumor samples of MDA-MB-231 tumors were from the mice and formalin-fixed and paraffin-embedded tumors were sectioned (5 m) and stained with hematoxylin and eosin.
Immunostaining for Ki67 was performed to evaluate intratumoral cell proliferation according to the manufacturer's protocol. Slides after staining were analyzed under an Eclipse TE200-U microscope (Nikon Instruments Inc., Melville, NY) as previously described 14 .
Immunostaining for Ki67 was performed to evaluate intratumoral cell proliferation according to the manufacturer's protocol. Slides after staining were analyzed under an Eclipse TE200-U microscope (Nikon Instruments Inc., Melville, NY) as previously described 14 .
8
Quantitative Wound Healing Assay with MDA-MB-231 Cells
9
Fibroblast Migration and Wound Healing
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Fibroblast migration was analyzed as previously described [74 (link)]. A wound was created through the cell monolayer using a 200 µL pipette tip. When required, cultured cells were then incubated with Panx1 channel blockers: 200 µM probenecid (PBN) (Thermo Fisher Scientific, Waltham, MA, USA), 100 µM 10Panx mimetic peptide, or scrambled mimetic peptide 10Panx (Sc 10Panx) 100 µM (Tocris, St. Louis, MO, USA). The wound areas were measured for 24 h, and standardized digital images were acquired every 2 h using a phase-contrast Nikon Eclipse TE200-U microscope (Nikon Instruments, Melville, NY, USA). Image analysis was performed using ImageJ (version 1.49v; NIH, MD, USA). Wound area = 100% − percentage of the initial wound area size.
The purinergic receptor antagonists used here were: 10 µM A-740003 (Tocris, MO, USA), 100 µM suramin (Sigma-Aldrich, St. Louis, MO, USA), 200 µM Brilliant Blue G (BBG, Sigma-Aldrich, Louis, MO, USA), 10 µM AR-C118925XX (Tocris, St. Louis, MO, USA), 5 µM MRS-2768 (Tocris, Louis, MO, USA), and 1 mM ATP (Sigma-Aldrich, Louis, MO, USA). Purinergic receptor antagonists and agonists were incubated for 30 min before inducing the wound and maintained for the experiment’s duration.
The purinergic receptor antagonists used here were: 10 µM A-740003 (Tocris, MO, USA), 100 µM suramin (Sigma-Aldrich, St. Louis, MO, USA), 200 µM Brilliant Blue G (BBG, Sigma-Aldrich, Louis, MO, USA), 10 µM AR-C118925XX (Tocris, St. Louis, MO, USA), 5 µM MRS-2768 (Tocris, Louis, MO, USA), and 1 mM ATP (Sigma-Aldrich, Louis, MO, USA). Purinergic receptor antagonists and agonists were incubated for 30 min before inducing the wound and maintained for the experiment’s duration.
10
Wound Healing Assay for mCRPC and PC-3 Cells
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The mCRPC CWR-R1ca and PC-3 cells were plated in sterile, 24-well, flat-bottom plates (3 replicates/group). Cells were allowed to form a subconfluent monolayer per well overnight. Wounds were inflected in each cell monolayer using a sterile pipette tip (200 µL). Media were then removed and, using sterile PBS, cells were washed twice. Cells were then incubated in 0.5% serum-containing culture media, to which various OC treatments were added. Cells were incubated for 24 h or until the vehicle control (VC) wells’ wound closed. The media were then removed and the cells washed with sterile, precooled PBS and fixed with absolute ethanol and stained with Giemsa. The healing of each wound was visualized at 0 and 24 h, or until the full closure of the VC wound, using Nikon ECLIPSE TE200U microscope (Nikon Instruments Inc., Melville, NY, USA). Digital images of each wound were captured and travel distance determined by comparing the wound width at 24 h or ending the experiments’ hours with the wound width at the start of treatment (zero time). The obtained % migration was calculated by setting the gap width at t0 as 100%. Each experiment was conducted in triplicate to confirm reproducibility.
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