The invasion of MDA-MB-231 cells or MDA-MB-157 cells was determined using a corning invasion chamber 24-well plate (354480, Corning BioCoat, USA). The membrane was coated with extracellular matrigel matrix (BD Biosciences, CA, USA) at 37 °C for 30 min. Then serum-free cell suspension (1 × 106/well) was placed into the upper chamber. The lower compartment was filled with L-15 medium adding with TGF-β1 (20 ng/mL) in the absence or presence of SFN, R-SFN, and S-SFN. After being cultured for 24 h, uninvaded cells were removed. The invading cells on the underside of the membrane were fixed with formaldehyde (4%) and stained with crystal violet (0.1%). Cells were visualized under a bright-field microscope (KEYENCE, Osaka, Japan).
Bright field microscope
Manufactured by Keyence
Sourced in Japan
The Bright-field microscope is a type of light microscope that illuminates the sample with bright, uniform light. It is used to observe and examine specimens by transmitting light through the sample. The core function of the Bright-field microscope is to magnify and provide a clear, high-contrast image of the specimen being observed.
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Lab products found in correlation
Murine tumor necrosis factor α, by Thermo Fisher Scientific (1 mentions)
Noggin, by R&D Systems (1 mentions)
R spondin 1, by R&D Systems (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
Polycarbonate filter, by Cytiva (1 mentions)
Jsm 7600f, by JEOL (1 mentions)
Freeze dryer, by Martin Christ (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Bz x800, by Keyence (1 mentions)
10 protocols using bright field microscope
1
Invasion Assay of Breast Cancer Cells
2
Rose Bengal Cell Staining Protocol
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The cells were washed three times with Ca2+/Mg2+ free PBS, and then were incubated in 0.1% rose bengal solution for 2–5 min followed by being washed three times in Ca2+/Mg2+-free PBS. Stained cells were imaged using the Keyence Brightfield microscope and the area of staining was quantified using Image J.
3
Intestinal Enteroid Generation and TNF-α-Induced Cell Death
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Enteroids were generated from isolated small intestinal crypts of β-actin cKO and control mice, as previously described (Lechuga et al., 2017 (link)). Briefly, mice were euthanized and their small intestinal segments were dissected, longitudinally opened, and washed with ice-cold PBS. Crypts were released using 30 min incubation with PBS containing 2 mM of EDTA at 4°C, with constant agitation, followed by mechanical shaking. Debris and villous fragments were discarded, and the resulting crypt fraction was collected by centrifugation and resuspended in growth factor reduced Matrigel (BD Bioscience). After Matrigel polymerization, DMEM/F12 medium containing HEPES, glutamine, N2 and B27 supplements, and growth factors [50 ng/ml epidermal growth factor, 500 ng/ml R-spondin 1, and 100 ng/ml Noggin (R&D Systems)] were added. Intestinal enteroids were allowed to differentiate for 7 days and were observed using a bright field microscope (Olympus BX41, Japan). Cell death was induced by treating enteroids with 100 ng/ml of murine tumor necrosis factor (TNF)-α (PeproTech, Cranbury, NJ) for 12 h. Viable and dead enteroids were distinguished by morphology, using a bright-field microscope (Keyence, Osaka, Japan) and counted. At least 50 enteroids per experimental group were examined. The percentage of dead enteroids was calculated from 3 independent experiments.
4
Matrigel Invasion Assay for Cell Motility
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Corning BioCoat Matrigel invasion chambers were used for invasion assays. Chambers were rehydrated in serum-free KSFM for 2 h at 37°C. Cells (105) resuspended in 500 µL of serum-free KSFM were seeded in the upper chamber, and 500 µL regular KSFM was added to the bottom of the well. Cells were incubated at 37°C for 24 h. After incubation, noninvading cells were removed from the upper surface with a cotton swab, and the remaining cells were fixed in 70% ethanol for 10 min at room temperature. Next, the invading cells were stained with 2% Crystal-Violet for 10 min at room temperature. Finally, the invading cell number was counted using a Keyence bright-field microscope at 10× with four fields per chamber. Invasion was calculated by the number of invaded cells per field. For 3D organotypic culture assays, invasion was determined by quantitating the area of invasive regions divided by the total epithelial length.
5
Visualizing Alginate Micro-structures Using Microscopy
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To visualize the TEP derived from the precursors (e.g. various alginate blocks), a bright-field microscope (Keyence, Japan) was employed. The fresh sample solutions were prepared prior to observation as described above. In order to visualize TEP with the bright-field microscope, precursor solutions were stained by freshly pre-filtered (0.05 μm polycarbonate filter) alcian blue solution as presented above. Stained samples were then observed under the microscope. For each sample, about 20 images were randomly taken.
The micro-structures of TEP derived from MG-, MM- and GG-blocks at different Na+/Ca2+ ratios were also observed by a field emission scanning electron microscopy (FESEM) (Jeol JSM-7600F, Japan). Although TEP were freeze dried prior to microscopic observation, this microscopic technique could still provide direct visualizations of evidence of TEP micro-structures. 10–50 mL of sample solutions prepared as described above were filtered through 0.1 μm polycarbonate filters (Whatman, United Kingdom) at a constant pressure of 0.2 bars and was then rinsed by 1 mL of Milli-Q water. Filters with retained alginate blocks were completely freeze-dried completely in a freeze dryer (Christ, Germany) for further examination. All samples were observed at least three times and 8–10 images were randomly recorded each time.
The micro-structures of TEP derived from MG-, MM- and GG-blocks at different Na+/Ca2+ ratios were also observed by a field emission scanning electron microscopy (FESEM) (Jeol JSM-7600F, Japan). Although TEP were freeze dried prior to microscopic observation, this microscopic technique could still provide direct visualizations of evidence of TEP micro-structures. 10–50 mL of sample solutions prepared as described above were filtered through 0.1 μm polycarbonate filters (Whatman, United Kingdom) at a constant pressure of 0.2 bars and was then rinsed by 1 mL of Milli-Q water. Filters with retained alginate blocks were completely freeze-dried completely in a freeze dryer (Christ, Germany) for further examination. All samples were observed at least three times and 8–10 images were randomly recorded each time.
6
Clonogenic Survival Assay after Radiation
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The HDS assay was carried out as described by Karasawa et al, with some modifications.27 (link)
Briefly, cells with about 50% to 60% confluence were irradiated, and the cells were kept in culture for an additional 3 days. Cells of each flask were then trypsinized and ×1/8 of cells for MDA-MB-231 and HCC1937 were plated onto new T25 flasks and subcultured further for 5 days. Eight days after exposure to radiation, cells were photographed with bright field microscope (Keyence, Osaka, Japan). Cells were then trypsinized, and the number of cells was counted with a hematocytometer. Outline of the experimental procedure for HDS assay after irradiation with olaparib treatment was summarized inFigure 1A .
Survival curves were fitted to the experimental data by regression analysis using the following linear quadratic equation28 (link)
:
where SF is the surviving fraction and D is radiation dose (Gy).
Briefly, cells with about 50% to 60% confluence were irradiated, and the cells were kept in culture for an additional 3 days. Cells of each flask were then trypsinized and ×1/8 of cells for MDA-MB-231 and HCC1937 were plated onto new T25 flasks and subcultured further for 5 days. Eight days after exposure to radiation, cells were photographed with bright field microscope (Keyence, Osaka, Japan). Cells were then trypsinized, and the number of cells was counted with a hematocytometer. Outline of the experimental procedure for HDS assay after irradiation with olaparib treatment was summarized in
Survival curves were fitted to the experimental data by regression analysis using the following linear quadratic equation28 (link)
:
where SF is the surviving fraction and D is radiation dose (Gy).
7
Immunohistochemical Analysis of Tumor Samples
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Tumor sections (4 μm) were analyzed using standard IHC techniques as per the manufacturer's recommendations (Vector Laboratories, Burlingame, CA, USA). Briefly, the paraffin‐embedded section on the slide was processed through three changes of xylene, followed by hydrating the tissue in a gradient of alcohol (100%, 95%, 90%, 80%, 70%, and 50% reagent alcohol with distilled water). The hydrated tissue was boiled in a pressure cooker with sodium citrate buffer at acidic pH for 20 min for antigen retrieval. The tissues were rinsed with 1X Tris buffer saline (TBS) and treated with hydrogen peroxide for 10 min at room temperature. The tissues were washed 2× with 1× TBS and incubated with anti‐mouse antibodies against CD31 (Cell Signaling Technologies, 1 : 200), or β‐catenin (Cell Signaling Technologies, 1 : 200) at 4 °C overnight, followed by incubating with anti‐rabbit ImmPRESS polymer reagent tagged with horseradish peroxidase or alkaline phosphatase (Vector Laboratories). Enzyme‐specific chromogen color development was performed using ImmPACT‐DAB or ImmPACT‐red to detect the bound primary antibodies. The cell nucleus was counterstained with hematoxylin (Vector Laboratories). The images were acquired using a brightfield microscope (Keyence, Itasca, IL, USA) at 200× magnification.
8
Murine Estrous Cycle Staging Protocol
9
Hematoxylin and Eosin Staining for Tissue Sections
10
Quantifying Optic Nerve Axon Survival
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After perfusion with 4% PFA in 0.1 M PBS, ONs were post-fixed with 2% glutaraldehyde and 2% PFA in 0.1 M PBS. Semi-thin (1 μm) cross sections of the ON 2 mm distal to the eye (globe) were collected. After staining with 1% paraphenylenediamine (PPD) in methanol:isopropanol (1:1), the semi-thin sections of the ON were imaged through a 100× oil immersion objective of a Keyence bright-field microscope tiled by 13 × 13 to cover the entire area of the ON. The stereological sub-sampling plugin AxonCounter45 (link) of NIH ImageJ was used to sample 5%–10% of the entire ON area with 10 × 10 μm counting frames. All surviving axons in sampled areas were manually identified and counted using ImageJ’s Cell Counter plugin. For SOHU glaucoma and EAE models, the mean of the surviving axon number in the injured ON was compared with that in the contralateral control ON to yield a percentage of axon survival value. The investigators who counted the axons were masked to the treatment of the samples.
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