The infectious titer of the virus stocks was determined in monolayers of Caco-2 cells grown in 96-well plates. For this, cells were infected as described above with serial two-fold dilutions of the virus stock, and after 16 h of incubation at 37 °C, the cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature (RT) and permeabilized by incubation with 2% Triton X-100 in PBS for 15 min at RT. The viral antigen was detected with serotype-specific polyclonal antibodies raised to the capsid spike protein of HAstV-1 (rabbit anti-spike1; dil. 1:1000), -2 (mouse anti-spike2; dil. 1:200) or -8 (rabbit anti-HAstV Yuc8; dil. 1:2000), as described [4 (link)], followed by incubation with the corresponding species-specific peroxidase-conjugated antibodies (KPL, Gaithersburg, MD, USA) diluted 1:3000. The focus forming units (ffus) were visually counted in in a Nikon TMS inverted phase-contrast microscope with a 20× objective.
Tms inverted phase contrast microscope
Manufactured by Nikon
Sourced in Canada, Japan
The TMS inverted phase-contrast microscope is a laboratory instrument designed for high-resolution imaging of specimens. It utilizes a phase-contrast technique to enhance the visibility of transparent samples, allowing for detailed observation and analysis.
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6 protocols using tms inverted phase contrast microscope
1
Determining Infectious Titer of Astrovirus
2
Cell Migration Imaging and Analysis
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For the examination of cell migration, the cells were seeded at a density of 3.5 × 105 on each cover slip (with and without the PP films), and after 8 h or 24 h of incubation, time-lapse imaging was performed using a Nikon TMS inverted-phase contrast microscope with an AmScope MU300 digital camera, capturing a series of 60 images over a duration of 30 min. To analyze the data, “ImageJ” was utilized to convert the captured images into specialized formats, while an open-source software, “Cell Tracker v1.1.1” ref. [22 (link)], facilitated the manual tracking of the migration parameters (the total way of run, the average distance from the origin, and the average speed) for each individual cell.
3
Wound Healing Assay for LN229 Cells
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LN229 cells were plated into wound healing inserts (Ibidi, Munich, Germany) at a density of 1.7×104 cells per insert well (7.7×104 cells/cm2) in DMEM supplemented with 10% FBS. Twenty four hours post-plating inserts were removed, wells washed with warm PBS and cells treated with H3, H6 or S100A4 in DMEM supplemented with 1% FBS for 8 h. Cells were fixed with 10% TCA for at least 1 h at 4°C and stained with SRB as described above. Images of the gap were captured using a TMS inverted phase contrast microscope (Nikon) with a DinoEye AM7023 eyepiece camera (AnMo Electronics, Taipei, Taiwan). Migration rate was evaluated as a percentage of the gap area covered by migrated cells using ImageJ software package (U. S. National Institutes of Health, Bethesda, Maryland, USA).
4
Bone Marrow Colony-Forming Assay
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A colony-forming unit assay was performed for bone marrow (BM) cells collected from both tibiae and femoral bones. The cells were plated in methyl cellulose medium (MethoCult M3231, Stemcell Technologies Inc., Vancouver, BC, Canada) at 2 × 105 cells/mL and incubated at 37°C and 5% CO2. After 12 days of incubation, the colonies were scored based on morphology as burst-forming unit-erythoid (BFU-E) under a Nikon TMS inverted phase contrast microscope (Nikon Instruments, Melville, NY).
5
Assessing Macrophage Morphological Changes
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J774A.1 mouse macrophage cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin as previously described [32 (link)]. Cells were grown at 37°C in a humidified 5% CO2 incubator. Cells were lifted by scraping and diluting ten-fold in complete growth medium. To assess the effect of each protein on cell morphology, confluent cells were diluted to 250000 cells/ml and mixed with 300 nM of toxin. From this cell suspension, 37500 cells were seeded in triplicate in a 96-well plate and incubated for 20 h. Cells were then assessed and imaged under a Nikon TMS inverted phase-contrast microscope with the 20× objective (Nikon Canada; Mississauga, Canada) to identify any morphological changes.
6
Rat Aorta Angiogenesis Bioassay
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The rat aorta angiogenesis bioassay [33 (link)] was modified to enhance the ability to screen for pro-angiogenic activity in the HPLC-derived fractions by reducing the concentration of heat inactivated foetal calf serum (HIFCS) from 20% to 5% and incubating for 7 days in 48 well culture plates (Costar, Corning, Lowell, MA) at 37°C in 5% CO2 [36 (link)]. Six technical and three biological replicates were used and the medium was changed on day 4 of culture. Vessel outgrowth was quantified as percent growth under a Nikon TMS inverted phase contrast microscope (Nikon Instrument Inc., Tokyo, Japan) at 40 × magnification on days 5, 6 and 7. Control cultures either received medium with the diluent alone or with the known anti-angiogenic agent, PI-88 [33 (link)] added at 100 μg/mL. Vessel outgrowth was determined as the percentage of a microscope field, outside the aorta ring, that was occupied by blood vessels using ImageJ software as previously described [40 (link)] or measured manually, particularly when large numbers of column fractions needed to be scored.
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