Approximately 700 cancer cells and 1,400 stellate cells were seeded in culture medium containing 1% FBS into the apical compartment of an Incucyte Clearview 96‐well plate (4582, Sartorius, Epsom, UK). Basolateral compartments were filled with culture medium containing 10% FBS, and plates were placed in an Incucyte S3 Imaging System (Sartorius). Images of the apical and basal sides of the 8‐μm porous membrane were captured every 4 h over 3 days. At each timepoint, migration was calculated as the percentage of cells present in the basal compartment compared to the total number of cells in both apical and basal compartments.
Incucyte s3 imaging system
Manufactured by Sartorius
Sourced in United States
The IncuCyte S3 imaging system is a live-cell analysis platform designed to capture and analyze real-time cellular processes within an incubated environment. It provides automated, non-invasive, and continuous monitoring of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (7 mentions)
Methotrexate, by Cayman Chemical (2 mentions)
Lometrexol hydrate, by Cayman Chemical (2 mentions)
Woundmaker, by Sartorius (1 mentions)
Imagelock 96 well plate, by Sartorius (1 mentions)
Incucyte 2019b rev2, by Sartorius (1 mentions)
Sytox green, by Sartorius (1 mentions)
Incucyte image analysis software, by Sartorius (1 mentions)
8 protocols using incucyte s3 imaging system
1
Cancer Cell Migration Assay
2
Live Cell Viability Imaging Assay
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Live cell viability was assessed using IncuCyte S3 imaging system (Sartorius). Briefly, cells were seeded in 96 well plate and treated with drug, either methotrexate (13960; Cayman Chemical Company) or lometrexol hydrate (18049; Cayman Chemical Company), in the presence of 1× Cytotox green reagent (ESS4633; Sartorius), a highly sensitive cyanine nucleic acid dye. Cells were imaged for 3 days with live cell imaging every 3h point. Dead cell quantification was performed using the IncuCyte software.
3
Live Cell Viability Assay
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Live cell viability was assessed using IncuCyte S3 imaging system (Sartorius). Briefly, an equal number of cells per condition were seeded in triplicates in a 96-well plate and treated with, methotrexate (Cayman Chemical Company, catalog no. 13960) or lometrexol hydrate (Cayman Chemical Company, catalog no. 18049), in the presence of the highly sensitive cyanine nucleic acid dye 1x green Cytotox reagent (Sartorius, catalog no. ESS4633) for SKMEL28 cells or 1x red Cytotox reagent (Sartorius, ESS4632) for Yumm5.2 cells. Cells were imaged for 3 days with live cell imaging every 3 hours. Dead cell quantification was performed using the IncuCyte software.
4
Wound Healing Assay in Hypoxia
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For migration assays, the cells were cultured for 24 h to confluence on ImageLock 96-well plates (Essen Bioscience) in normoxia and hypoxia. For experiments with parental MDA-MB-231 or 4T1 cells, 100 µM APCP was added to the culture medium. Scratch wounds were made with WoundMaker (Essen Bioscience), after which the wells were filled with fresh medium. Images were captured every 6 h for 24 h with the IncuCyte S3 imaging system (Essen Bioscience). Wound confluences were analyzed using IncuCyte 2018B software.
5
Time-lapse Microscopy of Cell Cultures
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Vehicle or indicated concentrations of drugs were added to cells (2500–5000 cells per well in a 96-well plate). Cells were imaged at 5 min to 1 h intervals for 5–10 days using IncuCyte S3 imaging system (Essen Bioscience, Michigan, USA) with a 20x objective. IncuCyte 2019B Rev2 software (Essen BioScience Michigan, USA) was used to calculate mean confluences (three parallel wells per treatment) from phase contrast images. For FUCCI-HeLa cells, fluorescence channels were used. Representative wells were selected for time-lapse movies, which were built with ImageJ ver 1.47d (25 (link)).
6
Real-Time Cell Death Monitoring of Activated BMDMs
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Real-time cell death assays were performed using an IncuCyte S3 imaging system (Essen Biosciences). BMDMs were seeded in 12-well plates (106 cells/well) and stimulated. After infection, 100 nM SYTOX Green (Thermo Fisher Scientific, S7020) was added. The images were acquired every 1 h at 37°C and 5% CO2. The resulting images were analyzed using the software package supplied with the IncuCyte imager, which counts the number of SYTOX Green-positive BMDM nuclei (Sytox+ BMDM nuclei) present in each image.
7
Real-Time Cell Death Monitoring of Activated BMDMs
8
Quantifying Cell Death Kinetics
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