Cells were seeded into 96-well tissue culture plate (Invitrogen Corporation) at the density of 2,000–10,000/ml. Cell growth was monitored by measuring confluence for 72–144 hours with IncuCyte ZOOM live-cell kinetic imaging system (Essen BioScience, Ann Arbor, MI). Cells were seeded into 96-well tissue culture plate at the density of 2,000–10,000/well. Thirty minutes after seeding, the plate was washed twice with 1X Dulbecco's Phosphate-Buffered Saline (DPBS; Life Technologies) and replaced with fresh complete medium. Initial images after seeding and images after medium replace were captured with the IncuCyte ZOOM live-cell kinetic imaging system. The percentages of the population of adhered cells were calculated by measuring confluence and compared between DU145 WT and lenti TGFBRII-gRNA/Cas9-treated cells. Cells were seeded into Image Locker 96-well tissue culture plate (Essen BioScience) at the density of 50,000/ml. Three hours later, scratch wound area was created by using Essen 96-well WoundMaker (Essen BioScience). Wound healing (cell migration) was monitored for 18 hours and images were captured with the IncuCyte ZOOM live-cell kinetic imaging system every 2 or 3 hours.
Incucyte zoom kinetic live cell imaging system
Manufactured by Sartorius
Sourced in United States
The IncuCyte Zoom Kinetic Live Cell Imaging System is a cell imaging system that allows for real-time, automated monitoring and analysis of living cells within a cell culture incubator. It provides time-lapse imaging and quantitative data on cell proliferation, migration, and other dynamic cellular processes.
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5 protocols using incucyte zoom kinetic live cell imaging system
1
Monitoring Cell Adhesion and Migration
2
Scratch Wound Assay in SW480 Cells
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Scratch wound assay was performed in SW480 cells, transfected with either pcDNA3.1/pIHV empty vector, respective wild-type or mutant constructs of B3GNT2, B4GALT2, and ST6GALNAC2, using the automated IncuCyte ZOOM live cell kinetic imaging system (Essen BioScience, Ann Arbor, MI) as per the manufacturer’s instructions over a period of 48hrs.
3
Quantitative Wound Healing Assay
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Cell migration was assessed by wound healing assay using an IncuCyte Zoom Kinetic Live Cell Imaging System (Essen BioScience). Ninety‐six‐well ImageLock Plates (Essen BioScience) were coated overnight with 300 μg/ml of 3‐D Culture Matrix Collagen I (Cat# 3447‐020‐01, R&D systems). The following day, collagen I dilution was aspirated, and cells were plated in six replicates to form fully confluent monolayers. Four hours after seeding, scratches were made with a 96‐pin WoundMaker (Essen Bioscience). Cells were covered in 100 μl of media. Migration was quantified using the Relative Wound Density metric calculated using IncuCyte software. Three separate assays were conducted.
4
Fibroblast Growth Dynamics Across Donors
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Fibroblasts from different donors [neonates with cleft lip with (n=2) or without (n=2) family history; children, n=2; adult facial (n=2)/breast (n=2) fibroblasts] were seeded at a density of 750 cells/well in 96-well plates. To achieve full adherence, cells were incubated at 37°C overnight in DMEM with 10% FBS. The next day, the culture medium was changed to media containing following active substances. TGF-β signaling blockade was performed selectively using small drug inhibitor SB-431542 (Sigma-Aldrich) at a concentration of 10 µmol/l, which completely abrogates phosphorylation of SMAD2 in fibroblasts (29 (link)). The control cells were maintained in DMEM plus 10% FBS. Recombinant human TGF-β1, TGF-β2 and TGF-β3 (all Sigma-Aldrich) were dissolved in 4 mM HCl with 0.1% of bovine serum albumin at a concentration 10 ng/µl and, for the experiments, they were used at a final concentration of 10 ng/ml DMEM. Cell proliferation was continuously monitored using an IncuCyte ZOOM Kinetic live cell imaging system (Essen BioScience, Ann Arbor, MI, USA) and measured as the phase object confluence percentage. All experiments were performed in triplicate and monitored for one week. Graphs were plotted and aligned (at 15% confluence) in the R statistical environment. The growth of cells was compared after 96 h, where the growth curves were in the log phase.
5
Apelin Peptide Proliferation Assay
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Twenty-four hours after cell seeding onto 96-well plates (3 × 103 cells), apelin peptides (100 nM) in MEM-α medium were added into the cells and incubated in IncuCyte ZOOM Kinetic Live Cell Imaging System (Essen Biosciences Inc., Ann Arbor, MI, USA) for 72 h. The images were taken every 24 h. The results are presented as a proliferation rate (percent of confluence in given time to percent of confluence in time zero). The experiments were performed three times and each independent experiment consisted of three measurements.
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