Cell viability on monolayered culture was measured via detection of cellular metabolic activity using a CellTiter-Glo® assay (Promega, Belgium). After siRNA-NG transfection and CAD treatment, the assay was performed following manufacturer's instructions. For spheroids, the cell viability was detected by microscopy imaging of visual alterations in the spheroid integrity and by performing CellTiter-Glo® 3D Cell Viability Assay (Promega, Belgium) according to the manufacturer's protocol after 20 h treatment with 5, 25, 50 µM of ebastine. Data are presented as percentage of viable cells calculated from the luminescence signal of each condition relatively to non-treated cells and by taking in account the background luminescence of the medium. Transmission images of spheroids were detected by a laser scanning confocal microscope (Nikon A1R HD confocal, Nikon, Japan), equipped with a 10× air objective lens (10× Plan Apo, NA 0.45, WD 4000μm, Nikon, Japan).
A1r hd confocal
Manufactured by Nikon
Sourced in Japan
The A1R HD confocal is a high-definition confocal microscope designed for advanced imaging applications. It features a high-resolution imaging sensor and sophisticated optics to capture detailed, high-quality images of samples. The core function of the A1R HD confocal is to provide advanced confocal imaging capabilities for research and analysis purposes.
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Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Nis elements software, by Nikon (2 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Apo 100 1.49 na objective, by Nikon (1 mentions)
Ti2 e, by Nikon (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
8 protocols using a1r hd confocal
1
Evaluating Cell Viability in 2D and 3D
2
Evaluating siRNA-Mediated Silencing in 3D Tumor Spheroids
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Tumor spheroids were seeded and allowed to grow for 3 days. After two consecutive washing steps with Opti-MEM, chol-siRNA was applied in Opti-MEM at the final concentration of 400 nM and cells were incubated at 37°C for 24 h. Subsequently, spheroids were washed with CCM, ebastine (35 µM) in CCM was applied for 20 h followed by 24 h incubation in fresh CCM.
For confocal imaging, spheroids were transferred to a glass-bottom 96-well plate (Grainer Bioone, Frickenhausen, Germany), and stained with Hoechst 33342 for 1 h (Molecular Probes, Erembodegem, Belgium). Imaging was performed with a laser scanning confocal microscope (Nikon A1R HD confocal, Nikon, Japan), equipped with a 10× air objective lens (10× Plan Apo, NA 0.45, WD 4000 μm, Nikon, Japan). The 409 nm and 488 nm lasers were applied to excite the DAPI labeled nuclei and the eGFP protein respectively. Fluorescence emission was detected through 450/50 nm (MHE57010) and 525/50 nm (MHE57030) filter cubes, respectively. Next, spheroids were prepared for flow cytometric detection as previously described and eGFP silencing data is presented as mean ± SEM of three independent spheroids.
For confocal imaging, spheroids were transferred to a glass-bottom 96-well plate (Grainer Bioone, Frickenhausen, Germany), and stained with Hoechst 33342 for 1 h (Molecular Probes, Erembodegem, Belgium). Imaging was performed with a laser scanning confocal microscope (Nikon A1R HD confocal, Nikon, Japan), equipped with a 10× air objective lens (10× Plan Apo, NA 0.45, WD 4000 μm, Nikon, Japan). The 409 nm and 488 nm lasers were applied to excite the DAPI labeled nuclei and the eGFP protein respectively. Fluorescence emission was detected through 450/50 nm (MHE57010) and 525/50 nm (MHE57030) filter cubes, respectively. Next, spheroids were prepared for flow cytometric detection as previously described and eGFP silencing data is presented as mean ± SEM of three independent spheroids.
3
Fluorescence Recovery After Photobleaching (FRAP) Analysis
4
Imaging Zebrafish Somitogenesis Mutants
5
Confocal Imaging and FRAP Analysis
6
Imaging Zebrafish Somitogenesis Mutants
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ProLong Gold antifade reagent (Life Technologies P36934) was used as mounting media. Imaging was performed by confocal microscopy on a Nikon A1R HD confocal on TiE microscope with a 100X NA 1.45 Plan Apo objective or 100X NA 1.49 TIRF Apo and resonant scanner, and sampled at Nyquist with a pinhole diameter of 75.35 μm and xy pixel size of 0.13 μm/px. Large tiled images were acquired to cover the whole PSM tissue of an embryo with 0.27 μm z-stack. Images were stitched with Nikon NIS-Elements software. In total, we imaged 24 wild-type, 18 her1ci301/+ her7hu2526/+, 28 her1ci301 her7hu2526, 24 her1b567/+ her7b567/+,14 wild-type sibling of her1b567/+ her7b567/+, 23 wild-type grown at 21.5°C, 23 wild-type grown at 28°C, 27 her1ci301/+ her7hu2526/+ gene-paired mutant (grown at 21.5°C), 37 her1ci302/+;her7+/hu2526 gene-unpaired mutant (grown at 21.5°C) embryos. Original microscopy image files are provided at the BioStudies Database 29 .
7
Mitochondrial Morphology Imaging
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To check mitochondrial morphology, MitoGFP transduced cells were grown on coverslips in a 6-well plate. Post-Hcy treatment, cells were fixed using the aforementioned fixing solution for 10 min at RT. After a wash with 1× PBS, cells were mounted on the slides using ProLong Gold mounting solution with DAPI (Invitrogen, USA). Images were captured by Nikon confocal A1R HD attached with Ti2-E (Nikon, Tokyo, Japan) using 60× Nikon objective (1.42 NA).
8
Visualizing Endothelial F-Actin Architecture
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F-actin staining of endothelial cells was performed using rhodamine labeled phalloidin. At first, HUVEC/TERT2 cells were grown on coverslips in a 6-well plate. After treatment, cells were fixed using previously mentioned fixing solutions for 10 min at RT. Then, one wash with 1× PBS was given. Thereafter, cells were permeabilized in 0.1% Triton X-100 in PBS for 5 min at RT to increase permeability. Samples were then washed twice in 1× PBS. Stock solution (400× in DMSO) of rhodamine phalloidin (Invitrogen) was diluted (2.5 μL in 1 mL of PBS) and then added to the samples followed by incubation in a cell culture incubator at 37 °C for 60 min. Then the samples were washed three times with 1× PBS. Finally, coverslips were mounted using Prolong Gold mounting solution with DAPI. Confocal images were acquired using Nikon confocal A1R HD attached with Ti2-E. Images were captured using a 100× Nikon objective (1.45 NA).
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