ESCC cells in RPMI-1640 with 10% FBS were seeded in 24-well plates (600 μL/well). The ESCC cells were incubated for up to 24 h until 100% confluency was achieved; thereafter, wounds were prepared using 1000-µL pipette tips. After washing with phosphate-buffered saline, wounded areas were captured using a CCD camera (40× magnification; Olympus, Tokyo, Japan). The medium was then changed to serum-free RPMI-1640 with/without rhIL-7 (20 ng/mL). In certain experiments, the ESCC cells were treated with either 10 μM LY294002 or 10 μM PD98059, and either a neutralizing antibody against IL-7R (1 μg/mL) or normal mouse IgG (1 μg/mL) as the negative control in the presence of rhIL-7 (20 ng/mL). After incubation for 24 h (for TE-11) or 48 h (for TE-9 and -10), each well was washed and the cells migrating to the wounded area were captured using a CCD camera (Olympus). We evaluated the migration ability by measuring the cell-free area with the polygon selection tool of ImageJ v1.52v (National Institutes of Health, Bethesda, MD, USA). Furthermore, the percentage wound coverage was compared between before treatment (Abefore) and after 24 or 48 h (Aafter); [(Abefore − Aafter)/Abefore] × 100. A schematic of the experimental procedure of the wound healing assay is shown in Figure S1C .
Ccd camera
Manufactured by Olympus
Sourced in Japan, Germany, United States
The CCD camera is a digital imaging device that captures light and converts it into electronic signals. It functions by using a charge-coupled device (CCD) sensor to detect and record images.
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Lab products found in correlation
Ix50 fluorescent microscope, by Olympus (8 mentions)
Bx51 microscope, by Olympus (5 mentions)
Dab kit, by Vector Laboratories (4 mentions)
Axiovert, by Zeiss (3 mentions)
Spark 10m fluorescent microplate reader, by Tecan (3 mentions)
Ix71 microscope, by Olympus (3 mentions)
Jem 1230, by JEOL (3 mentions)
Bx53 microscope, by Olympus (3 mentions)
Item software, by Olympus (2 mentions)
Cellstain double staining kit, by Dojindo Laboratories (2 mentions)
Ix71s1 f 2, by Olympus (2 mentions)
Lab 6 cathode, by Olympus (2 mentions)
Nucblue live cell stain, by Thermo Fisher Scientific (2 mentions)
H4 100, by Olympus (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Jem 1400 electron microscope, by JEOL (2 mentions)
Kh2po4, by Merck Group (2 mentions)
Na2hpo4, by Merck Group (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
126 protocols using ccd camera
1
Wound Healing Assay with ESCC Cells
2
Quantitative Microscopy Analyses of Cellular ROS
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Morphometrical analysis was performed using high power optical fields digitized with a CCD camera (Soft Imaging System) equipped to a BX51 microscope (Olympus) and imaging Software (analySIS, Soft Imaging System). Fluorescence-labeled cells as well as PI staining intensities were analyzed by an Olympus microscope (IX 70) equipped with a TRITC (excitation filter 520–550 nm, barrier filter 580 nm) and FITC (excitation filter 450–490 nm, band filter 520–550) narrow band filter, a CCD camera and image software. Cross talk and fluochrome filter leakage between channels was controlled and corrected using software (Lv3.1). For detection of intracellular peroxides, intracellular accumulation of reactive oxygen species (ROS) was visualized by 5-carboxy-2-dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes, Invitrogen) for 30 min at 37°C. Fluorescence measurements were performed on a fluorescence plate-reader at Ex λ 488 nm and Em λ 530 nm. Cell cycle analysis was performed on a FACS Calibur (BD Biosciences) equipped with an argon laser line and a red laser diode.
3
Zebrafish Ultrastructural Analysis
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Zebrafish (adult; ∼25 mm standard length) were anesthetized with MS-222 and cut into small pieces. The specimens were then fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer for 60 minutes at room temperature. The fish pieces were then treated with 2% osmium in 0.1 M cacodylate buffer containing 4% sucrose for 60 minutes at room temperature. Then, samples were dehydrated through a graded ethanol series and embedded in Quetol-812 resin. Ultrathin sections were obtained with an Ultramicrotome (Reichert-Jung Ultracut E), counterstained with uranyl acetate and lead citrate, and examined on a transmission electron microscope (JEM-1200 EX, JEOL, Japan) with an acceleration voltage of 80 or 90 kV. Micrographs were recorded with a CCD camera (Morada) using the iTEM software (Olympus Soft Imaging Solutions).
4
Ultrastructural Analysis of Late Embryos
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Late stage embryos of the desired genotype were prepared for TEM analysis essentially as described [65 (link)]. Following washing with 0.1 M cacodylate buffer, tissue was stained with 1% osmium tetroxide for 1 hour, dehydrated in a graded ethanol series and propylene oxide and embedded in Poly/BedR 812 (Polysciences, Inc., Eppelheim, Germany). Semi-thin cross sections of each 1 μm were made on a Leica Ultracut S ultramicrotome. Sections were stained with toluidine blue for 3 minutes at 70°C. Image acquisition was on a Zeiss Axiophot microscope system. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a FEI Morgagni electron microscope. Digital images were taken with a Morada CCD camera and the iTEM software (Olympus Soft Imaging Solutions GmbH, Münster, Germany).
5
Transmission Electron Microscopy of HPDL Cells
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TEM analysis of HPDL cells was performed on the basis of a previous study [64 (link)]. Briefly, HPDL cells cultured on plates were fixed with 2% glutaraldehyde in PBS at 4°C overnight, and then with 2% tetraphosphate osmium at 4°C for 1 hour. Then, HPDL cells were dehydrated with 50%, 70%, 90% and 100% ethanol solutions, embedded in Quetol-812 (Nisshin EM, Tokyo, Japan), and polymerized at 60°C for 48 hours. Ultrathin sections cut enface at 70 nm thicknesses were collected on diamond knifes and placed on copper grids. They were stained with 2% uranyl acetate at R/T for 15 min, and rinsed with distilled water, and then stained with Lead stain solution (Sigma-Aldrich Co., MO, USA) at R/T for 3 min. The grids were observed under a transmission electron microscope (JEM-1400 plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 80 kV. Digital images were captured with a CCD camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Slice preparation and imaging analysis were performed in accordance with the protocols of Tokai Electron Microscopy, Inc. (Aichi, Japan).
6
Quantifying Cell Colony Growth
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Phase-contrast photographs of cells were obtained 1, 4, 7, 14, 21, and 28 days after plating using a phase-contrast microscope equipped with a CCD camera (Olympus Optical Co., Tokyo, Japan). The growth of some colonies was pursued and digitally recorded. At least 10 fields per dish were randomly selected and 3 dishes or wells were used per experiment. At least three independent experiments were completed. All captured images were processed using specialized software (Olympus cellSens Dimension Desktop 1.12). The number of colonies, the number of cells per colony, and the size of the cells and colonies were measured.
7
Evaluating Islet Viability via PI Staining
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The viability of the islets was determined by staining with propidium iodide (PI; Molecular Probes, Eugene, OR). PI-positive islets cells were defined as dead cells and counted by visual inspection of PI staining under a fluorescent microscope equipped with a CCD camera (Olympus Optical, Tokyo, Japan). Islets were scored mainly by evaluating the ratio of PI positive cells ( • ▶ Fig. 1a). The area of PI positive cells in each islets was measured by Image J software (https://imagej.nih.gov/ij/). According to the ratio of PI positive cells, scores from 10 to 0 were assigned to each islet: score 10 for less than 10 %, score 8 for less than 30 %, score 6 for less than 50 %, and scores 4-0 for over 50 %. In determining the scores 4, 2, and 0, morphological feature of the islets, i. e., round shape, collapsing, or disrupted, was considered in addition to the PI positive ratio ( > 50 %). Thus, the ratio of PI-positive cells and the morphological changes of the islets were included in our original index to evaluate viability ( • ▶ Fig. 1a). To evaluate the score of islets, more than 10 islets were randomly picked for each evaluation and more than 2 examiners judged the score.
8
Ultrastructural Analysis of Cells by TEM
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For transmission electron microscopy analysis, the cells were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer, rinsed in 0.1 M cacodylate buffer, post-fixed for 1h in 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 M cacodylate buffer to enhance the staining of membranes. Cells were then rinsed in distilled water, dehydrated in alcohols and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope mounted with a Morada Olympus CCD camera.
9
TEM Ultrastructural Cell Analysis
10
Cell Viability Evaluation Using Fluorescent Stains
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Cellstain Double Staining Kit (Dojindo, Tokyo, Japan) was used to test cell viability as described previously [9 (link)]. Briefly, seeded cells were stimulated according to specific conditions. Afterward, propidium (4 μM) [12 (link)] and calcein-AM (2 μM) were used to stain cells. Ten minutes later, the live and dead cells were discriminated. The living cells hydrolyze calcein-AM by intracellular esterase, generating green fluorescence. In contrast, the dead cells showing red fluorescence. The cell images were recorded with an Olympus CCD camera attached to the immunofluorescent microscope (IX71S1 F-2; Olympus, Tokyo) under the identical settings.
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