The CPP apparatus consisted of two equal-sized compartments (30 cm long × 30 cm wide × 30 cm high), including one having a white interior and the other having a black interior that separated by a wall with a sliding door. Bio-Rad Gradient PCR (Bio-Rad Laboratories, Inc., Hercules, CA, USA); Agilent 2200 Bioanalyzer; Stratagene Mx3005P Real-time polymerase chain reaction (PCR) instrument (Agilent Technologies, Inc., Santa Clara, CA, USA); Agilent 2200 TapeStation (Agilent Technologies, Inc.); DU800 Nucleic Acid/Protein Analyzer (Beckman Coulter, Inc., Brea, CA, USA); IX53 Fluorescence Inverted Microscope (Olympus Corporation, Tokyo, Japan); Qubit 2.0 (Thermo Fisher Scientific, Inc.); Hiseq 2500 (Illumina, Inc., San Diego, CA, USA) and ND-1000 Nanodrop (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
Ix53 inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The IX53 inverted fluorescence microscope is a high-performance imaging system designed for a variety of cell and tissue-based applications. It features a stable and precise optical system, advanced illumination, and a range of modular accessories to support diverse research needs.
Automatically generated - may contain errors
Lab products found in correlation
Du800 nucleic acid protein analyzer, by Beckman Coulter (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Agilent 2200 tapestation, by Agilent Technologies (2 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Real time pcr mx3005p, by Agilent Technologies (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fitc labeled secondarygoat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Eclipse ti confocal microscope, by Nikon (1 mentions)
Transwell cell chamber, by Corning (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Six well plate, by Corning (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Cx31 upright microscope, by Olympus (1 mentions)
11 protocols using ix53 inverted fluorescence microscope
1
Multimodal Behavioral Characterization Platform
2
CPP Instrument for Behavioral Studies
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The CPP instrument was purchased from Shanghai Yu Yan Scientific Instruments Co., Ltd. (Shanghai, China). It is comprised of two equal-sized compartments (30 cm long × 30 cm wide × 30 cm high), one having a white inner part and the other having a black inner part, divided by a wall with a sliding door. Commercial equipment used here was as follows: Agilent 2200 Bioanalyzer, Stratagene Mx3005P Real-time PCR (polymerase chain reaction) instrument (Agilent Technologies, Paro Alto, California, USA); Bio-Rad Gradient PCR (Bio-Rad, Hercules, California, USA); DU800 Nucleic Acid/Protein Analyzer (Beckman, Pasadena, California, USA); IX53 Fluorescence Inverted Microscope (Olympus, Tokyo, Japan); Agilent 2200 TapeStation (Agilent Technologies, Paro Alto, California, USA); ND-1000 Nanodrop (Thermo Fisher, Waltham, Massachusetts, USA); Qubit 2.0 (Life Technologies, Carlsbad, California, USA); and Hiseq 2500 (Illumina company, Santiago, California, USA).
3
Hyphal Production in Candida Biofilms
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To study the effect of IP6 on hyphal production, 72-h dual-species biofilms were prepared and treated with 0.16% IP6 as described above. After treatment, the medium was retrieved, and the biofilm was washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 1 h. The wells were washed with PBS and stained with 0.2% calcofluor white for 30 min at room temperature. Images were obtained using an Olympus IX53 fluorescence inverted microscope. The proportion of hyphae relative to total Candida fungal units (yeast and hyphae) was determined using ImageJ software version 1.48.
4
Caco-2 Cell Viability Assay
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Caco-2
cells with good growth status were adjusted to a cell suspension of
8 × 104 cells/mL, inoculated uniformly at a volume
of 1 mL per well in 24-well cell culture plates, and cultured at 37
°C for 24 h. Then, the culture medium was aspirated, the cells
were washed twice with PBS, and the cell culture medium containing
liposomes with different contents of 0, 25, 50, 100, and 200 μg/mL
of Zanthoxylum alkylamides was added into each well.
After 6 h of culture, the morphology of the cells was observed under
an IX53 inverted fluorescence microscope (Olympus Corporation, Tokyo,
Japan).
cells with good growth status were adjusted to a cell suspension of
8 × 104 cells/mL, inoculated uniformly at a volume
of 1 mL per well in 24-well cell culture plates, and cultured at 37
°C for 24 h. Then, the culture medium was aspirated, the cells
were washed twice with PBS, and the cell culture medium containing
liposomes with different contents of 0, 25, 50, 100, and 200 μg/mL
of Zanthoxylum alkylamides was added into each well.
After 6 h of culture, the morphology of the cells was observed under
an IX53 inverted fluorescence microscope (Olympus Corporation, Tokyo,
Japan).
5
Immunofluorescence Characterization of Differentiated Cells
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Differentiated cells derived from hESCs were washed two times with 1XPBS and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer saline (pH 7.4) (Santa Cruz Biotechnology, USA) for 20 min. The cells were permeabilized for 15 min with 0.5% Triton X-100 (Sigma, USA) in PBS (PBST), and blocked for at least 1 hour with 6% bovine serum albumin (BSA) in PBST at room temperature. Primary antibodies, including mouse anti-NKX6.1 (1:2000; DSHB; F55A12), guinea pig anti-PDX1 (1:1000; Abcam, Ab47308), and rat anti- Insulin (1:1000; GN-ID4, DSHB), goat anti-Glucagon (1:3000; sc-7781; SantaCruz) were added to the cells and incubated overnight at 4 °C. The next day, primary antibodies were removed, and 0.5% Tween 20 (TBST) was used for washing the cells 3 times. Subsequently, cells were incubated with the appropriate 488 and 568 Alexa Fluor secondary antibodies (Thermo Fisher Scientific) with a dilution of (1:500). Nuclear staining was examined after counterstain with Hoechst 33342 (1 μg/ml) (Thermo Fisher Scientific). The cells were visualized by Olympus IX53 inverted fluorescence microscope (Japan).
6
PEDV Infection of HEK293 Cells
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The HEK293 monolayers were grown to 100% confluency in 24-well plates and incubated with dimethyl sulfoxide (DMSO) or with different concentrations of the p53 activator Nutlin-3 (10, 2.5 μM). After 24 h, HEK293 cells were infected with PEDV at a multiplicity of infection (MOI) of 1. At 24 hpi, cells were fixed with 33.3% acetone and stained with PEDV-S antibody (1:200 dilution), followed by a FITC-labeled secondary goat anti-mouse IgG (1:400 dilution) (Jackson Immunoresearch, West Grove, USA). An Olympus IX53 inverted fluorescence microscope was used for visualization.
7
Time-lapse Microscopy of Bacterial Cells
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Phase contrast and fluorescence microscopy was performed using bacteria spotted on agarose pads, as previously described (Roghanian et al., 2019 (link)). For still images, bacteria were grown to late log phase in CDM containing 100 µM IPTG, diluted in PBS and transferred to agaros pads. Microscopy was performed using Olympus IX53 inverted fluorescence microscope, and the images were analyzed using the software Olympus cellSens Dimensions 1.18. For time-lapse microscopy, bacteria were scraped from o/n chocolate agar plates with 30 µM kanamycin, diluted in PBS and 2 µl were spotted on agarose pads containing CDM with 100 µM IPTG. The samples were transferred to the microscope enclosure and allowed to adjust to 37 °C for 30 min. A Zeiss Axio Observer.Z1 inverted light microscope with a motorized stage was used for this purpose as previously described (Frojd and Flardh, 2019 (link)). Zeiss ZEN 2.3 software was used for analysis of the images. Phase contrast and fluorescence images were recorded every 10 min for up to 6 h at 37 °C in temperature-controlled settings. All images were taken with the 100x objective in phase-contrast and fluorescent channels.
8
Wheat Germ Agglutinin Staining for Cardiomyocyte Morphometry
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Wheat germ agglutinin (WGA) staining was performed for cardiomyocyte circumference measurement as described previously [28] . Briefly, after deparaffinization, cardiac tissue (n=3 each sham group, n=6 each TAC group) rehydrated through serial concentration of ethanol. Antigen retrieval was performed using acetate-based retrieval solution followed by blocking the heart sections using animal-free blocking buffer (Cell signaling #15019). Sections were incubated with WGA-Alexa Fluor 549 (5μg/mL) dye for 15 minutes at room temperature followed by 3X washing with PBS and mounting using mounting media. To analyze the role of NRK-2 in Ang II-induced hypertrophy in AC16 cardiomyocytes, an equal number of cells were seeded on the coverslip and transfected with NRK-2 or control plasmids as described above. Post-transfection, cells were treated with 1.0 μM Ang II or vehicle for 48 hours. After treatment, coverslips were washed with PBS and stained with WGA for 15 minutes at room temperature. Imaging was done using Olympus IX53 Inverted fluorescence microscope with a 40X object. Representative images were acquired under Nikon Eclipse Ti confocal microscope. Cell circumference was measured using NIS Element software. The experiment was repeated twice, in duplicate and triplicate using cells from two different passages.
9
Transwell Assay for HUVEC Migration
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HUVECs were resuspended in serum-free medium, and 1.0 × 104 cells were loaded into 8.0-μm Transwell cell chambers (Corning, U.S.A.). According to the HUVEC experimental grouping, CM or HG medium (serum-free) were added to the lower chambers. After 12 h of incubation at 37°C and 5% CO2, the culture medium was extracted from the Transwell lower chambers in each HUVEC group and centrifuged for 5 min at 800 rpm. The cells were resuspended in 0.01 mmol/l PBS, and the number of cells in the suspension was counted. The non-migrated cells were removed from the Transwell chambers, and the cells were washed three times with 0.01 mmol/l PBS. HUVECs that migrated to the other side of the membrane were fixed with 4% paraformaldehyde (Sigma, U.S.A.) for 2 h and stained with 1% Crystal Violet (Sigma, U.S.A.). Cell migration was observed with an IX53 inverted fluorescence microscope (Olympus, Tokyo, Japan), and the cell migration rate was calculated at 12 h as follows: [(cells in Transwell cell chamber for 12 h + cells in heavy suspension)/1.0 × 104] × 100% [27 (link)]. Each batch of HUVECs was inoculated in ten wells with three replicates.
10
Evaluate HUVEC Wound Healing Dynamics
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HUVECs were plated in six-well plates (Corning, NY, U.S.A.) at 1 × 106 cells/well. When cells covered the bottom of the well, a central scratch was created by scraping cells away with a p1000 pipette tip, and the stripped cells were washed three times with 0.01 mmol/l PBS. According to the HUVEC experimental grouping, the HUVECs were cultured in HG medium or corresponding CM with 2 mmol/l hydroxyurea (cat. no. H8627; Sigma, U.S.A.) and incubated at 37°C in 5% CO2 for 24 h. Then, the cells were rinsed three times with 0.01 mmol/l PBS. The HUVECs were fixed with 4% paraformaldehyde (cat. no. 16005; Sigma, U.S.A.) at room temperature for 2 h and then stained with 1% Crystal Violet (cat. no. C6158; Sigma, U.S.A.). The scratches were recorded with an IX53 inverted fluorescence microscope (Olympus, Tokyo, Japan), and the cell scratch areas at 0 and 24 h were measured using Image-Pro Plus (IPP) 6.0.1 software (Media Cybernetics, Rockville, MD, U.S.A.). The residual rate of the scratch area at 24 h was calculated as follows: (cell scratch area at 24 h/cell scratch area at 0 h) × 100% [26 (link)]. Each batch of HUVECs was inoculated in ten wells with three replicates.
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