Virus titers were determined using a BacPAK™ Baculovirus Rapid Titer Kit (Clontech) according to the manual (https://www.takarabio.com/documents/User%20Manual/PT3153/PT3153-1_072213.pdf , accessed on 16 April 2022). The foci of infection (clusters of infected cells) were counted in duplicate wells using an inversion microscope (CKX31SF, Olympus, Tokyo, Japan). Virus titers (IFU/mL) were calculated by multiplying the average number of foci per well by the corresponding dilution factors.
Ckx31sf
Manufactured by Olympus
Sourced in Japan
The CKX31SF is an inverted microscope designed for routine cell culture observation and basic microscopy applications. It features a stable frame, a siedentopf binocular eyepiece, and a condenser lens system for bright-field illumination.
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11 protocols using ckx31sf
1
Baculovirus Rapid Titer Assay
2
Morphological Changes in Dermal Fibroblasts Exposed to W-NPs
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To determine by qualitative means the influence of W-NPs on morphological changes in BJ dermal fibroblasts, images of the cells exposed to different concentrations of W-NPs were obtained using an inverted optical microscope (CKX31SF, Olympus, Japan).
SEM was used to obtain complementary information both on cellular morphology changes induced after cells exposure to W-NPs and on prospective internalization of these nanoparticles under the BJ cell membrane surface. Briefly, the BJ cells were seeded onto coverslips and incubated for 24 h to adhere, followed by exposure to W-NPs for another 24 h. Then, the samples were fixed in 2.5% glutaraldehyde in PBS, followed by dehydration with ascending series of water-ethanol solutions (10, 30, 50, 70, 90 and 100% for 10 min each) and HMDS drying (100% for 10 min). After that, HMDS was decanted, and the samples were left under a hood to air-dry at room temperature. Afterward, the samples were coated with a thin layer of 5 nm of gold.
The SEM investigations on cellular morphology were performed using a Zeiss EVO 50 XVP equipment with LaB6 electron gun, in the secondary electron mode. The cell morphology after WNPs exposure was also investigated without the thin layer of gold, in order not to alter the morphological properties. These images are added as Supplementary Material.
SEM was used to obtain complementary information both on cellular morphology changes induced after cells exposure to W-NPs and on prospective internalization of these nanoparticles under the BJ cell membrane surface. Briefly, the BJ cells were seeded onto coverslips and incubated for 24 h to adhere, followed by exposure to W-NPs for another 24 h. Then, the samples were fixed in 2.5% glutaraldehyde in PBS, followed by dehydration with ascending series of water-ethanol solutions (10, 30, 50, 70, 90 and 100% for 10 min each) and HMDS drying (100% for 10 min). After that, HMDS was decanted, and the samples were left under a hood to air-dry at room temperature. Afterward, the samples were coated with a thin layer of 5 nm of gold.
The SEM investigations on cellular morphology were performed using a Zeiss EVO 50 XVP equipment with LaB6 electron gun, in the secondary electron mode. The cell morphology after WNPs exposure was also investigated without the thin layer of gold, in order not to alter the morphological properties. These images are added as Supplementary Material.
3
Osteogenic Differentiation Dynamics of hPSCs
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When grown into 80% confluence, hPSCs on the Matrigel surface were transferred into osteogenic medium (OM) that consisted of αMEM medium, 15% FBS, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin/streptavidin, 5 μg mL− 1 ascorbic acid, 10 mM sodium glycerophosphate and 10− 8 M dexamethasone. The OM was changed freshly every 2 days for 35 days. After induction for different times (0 days, 3 days, 7 days, 14 days, 21 days, 28 days and 35 days), the cells were observed using a phase-contrast microscope (CKX31SF, Olympus, Japan) with a CCD camera (MP3.3-RTV, Olympus, Japan), and their viability was detected using the cell counting kit-8 reagent.
4
Silk-VM Organoid Differentiation Protocol
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After three days, differentiation medium consisting of 1:1 DMEM/F12:Neurobasal medium, 1:100 N2 supplement, 10 μM SB431542, 150 ng/mL rhNoggin, 400 ng/mL SHH-C24II, and 1.5 μM CHIR99021 was added from day 0 to 10, following the same protocol used for generating conventional VM organoids. At day 10, the resulting 3D structures were mechanically detached from the bottom of the plate with a spatula and transferred to a 6-well plate (Corning, #3471) and grown in suspension (free floating). Silk-VM organoids were embedded in 30 µL droplets of Matrigel and cultured following the VM organoid-differentiation protocol described in the subsection “hPSC VM organoid differentiation”. Images were collected on phase-contrast inverted microscope (Olympus, #CKX31SF). Morphological classification (spherical/nonspherical) was performed in triplicate. Roundness measurements were based on bright-field images and calculated as the ratio between diameters of the largest inscribed and the smallest circumscribed circle of the organoid silhouettes (dotted line). Images were analyzed in ImageJ (NIH).
5
Evaluating Cell Migration after miR-4796 Transfection
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The cells were cultivated for 24 h after transfection with miR-4796 and treatment with OPL, and the transfected cells were washed twice with PBS. Then, the cells were lined with 1 mL pipette tips, followed by the removal of the exfoliated cell residue with PBS. The cells continued to be cultured for 24 h under conventional conditions before taking images by microscope (CKX31SF, Olympus Co., Ltd., Tokyo, Japan). The Image J (National Institutes of Health, Bethesda, MD, USA) software was used to analyze the distance between 0 h and 24 h cell scratch boundary. The cell spacing of 24 h was subtracted from the cell spacing of 0 h to obtain the migration distance of the cells cultured for 24 h.
6
Cell Clonogenic Assay for CC Cells
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There were three experimental groups of CC cell lines for this assay: i) Ctrl, siRNA negative control; ii) siRNA#1 transfection; and iii) siRNA#1 transfection combined with cisplatin (3 μg/ml; Qilu Pharmaceutical Co., Ltd.). At 48 h after transfection, 500 cells per well were added; three wells were used in each group. The cells were stained with crystal violet solution for 30 min and observed under an inverted microscope (CKX31SF; Olympus Corporation) at ×100 magnification 7-10 days later. The cell clones with >50 cells were counted as one monoclone, and the mean number of clones on the plate was used to calculate the cell clone-forming ability.
7
Liver Histology: Formalin Fixation
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Liver pieces were fixed in 3.7–4.0% neutral formalin (pH 7.4) for 24 h, dehydrated with 70% ethanol, and embedded in paraffin. Five μm thick sections were deparaffinized and stained with hematoxylin-eosin (H&E). Liver images (magnification 40X) were taken with an Olympus microscope (CKX31SF, Olympus Corp., Tokyo, Japan) coupled to an Olympus C-5060 camera (Olympus Corp., Tokyo, Japan).
8
Proliferation Analysis of UC-MSCs
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For proliferation analysis, UC-MSCs were passage cultured in StemMACS™ MSC Expansion Media Kit XF (Miltenyi Biotec, Germany). Cells were visualized using an Olympus CKX31SF inverted microscope (4× objective lens) and images were captured (three fields of view per replicate; three replicates). Cell number was assessed and compared using automated cell identification methods. This method already is demonstrated that it was sensitive enough to accurately detect both more minor changes in cell numbers and a more comprehensive range of cellular densities than spectrophotometric analysis of crystal violet-stained cells [24 (link)]. According to actual conditions, afore macro was changed:
9
Osteogenic Differentiation of hPSCs
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When grown into 80 % con uence, hPSCs on the Matrigel surface were transferred into osteogenic medium (OM) that consisted of αMEM medium, 15 % FBS, 1 % NEAA, 0.1 mM β-mercaptoethanol, 1 % penicillin/streptavidin, 5 µg•mL -1 ascorbic acid, 10 mM sodium glycerophosphate and 10 -8 M dexamethasone. The OM was changed freshly every 2 days for 35 days. After induction for different times (0 days, 3 days, 7 days, 14 days, 21 days, 28 days, and 35 days), the cells were observed using a phase-contrast microscope (CKX31SF, Olympus, Japan) with a CCD camera (MP3.3-RTV, Olympus, Japan), and their viability was detected using the cell counting kit-8 reagent.
10
Osteogenic Differentiation of hPSCs
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Loading [MathJax]/jax/output/CommonHTML/jax.js When grown into 80% con uence, hPSCs on Matrigel surface were transferred into osteogenic medium (OM) that composed of αMEM medium, 15% FBS, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin/streptavidin, 5 µg•mL - 1 ascorbic acid, 10 mM sodium glycerophosphate and 10 - 8 M dexamethasone. The OM was changed freshly every 2 days for 35 days. After induction for different times (0 day, 3 days, 7 days, 14 days, 21 days, 28 days, 35 days), cells were observed using a phasecontrast microscope (CKX31SF, Olympus, Japan) with a CCD camera (MP3.3-RTV, Olympus, Japan), and their viability was detected using a cell counting kit-8 reagent.
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