First, 4 h after transfection, 8000 endothelial cells were placed into each well of a 96-well ImageLock plate (Essen Biosciences, Ann Arbor, MI, USA) in HUVEC media. The cells were placed into the incubator at 37 °C, 5% CO2 for 48 h. A ‘Woundmaker 96’ (Essen Biosciences) was used to create the wounds in this experiment. The ImageLock plate was inserted into an Incucyte incubator at 37 °C, 5% CO2, which was programmed to take images at regular intervals using IncuCyteZoom 2015A software (Essen Biosciences). The images were downloaded and analysed using ImageJ. All experiments were run in triplicate.
Incucyte incubator
Manufactured by Sartorius
Sourced in Germany
The Incucyte incubator is a cell imaging system designed for real-time, long-term monitoring of cell cultures. It provides a controlled environment for cell growth and allows for automated, non-invasive imaging of cells over an extended period.
Automatically generated - may contain errors
4 protocols using incucyte incubator
1
Endothelial Cell Wound Healing Assay
2
Angiogenesis Assay in 3D Matrigel
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First, 48 h after transfection, 70 µL of Matrigel (Corning, Corning, NY, USA) was placed into a well of a 12-well plate and allowed to solidify by placing the plate into the incubator at 37 °C, 5% CO2 for 30 min. Cells were diluted to a concentration of 140,000 cells per ml in HUVEC media and 1 mL of cell solution was added to the Matrigel well. The plate was uploaded into an IncuCyte incubator (Essen BioSciences) at 37 °C, 5% CO2, which was programmed to take pictures at regular intervals using IncuCyteZoom2015A software (Essen BioSciences). The images were downloaded and analysed using the online available software ImageJ. All experiments were run in triplicate.
3
Automated Wound Healing Assay
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Approximately
25 000 cells
per well were seeded on Sartorius 96-well LockView or ImageLock microplates
(Sartorius AG) the night prior to wound formation. The cell monolayer
was scratched with the IncuCyte WoundMaker that simultaneously makes
equivalently sized scratch wounds in the monolayer in all wells, and
the medium was replaced with a compound-containing medium supplemented
with 1% fetal bovine serum (FBS) to induce cell migration and reduce
cell proliferation. Serial dilutions (1 μL) of the compounds
in DMSO were added to obtain the 1 nM to 10 μM final concentrations,
and the plate was mixed gently. The plates were placed in the IncuCyte
incubator and imaged in a bright field with a 10× objective every
3 h for 48 h. Analysis of the wound confluence and width was performed
with an automated script in IncuCyte 2021C software provided by Sartorius.
25 000 cells
per well were seeded on Sartorius 96-well LockView or ImageLock microplates
(Sartorius AG) the night prior to wound formation. The cell monolayer
was scratched with the IncuCyte WoundMaker that simultaneously makes
equivalently sized scratch wounds in the monolayer in all wells, and
the medium was replaced with a compound-containing medium supplemented
with 1% fetal bovine serum (FBS) to induce cell migration and reduce
cell proliferation. Serial dilutions (1 μL) of the compounds
in DMSO were added to obtain the 1 nM to 10 μM final concentrations,
and the plate was mixed gently. The plates were placed in the IncuCyte
incubator and imaged in a bright field with a 10× objective every
3 h for 48 h. Analysis of the wound confluence and width was performed
with an automated script in IncuCyte 2021C software provided by Sartorius.
4
Time-lapse Imaging of CF BCs
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BCs from CF donor 1288insA/406-1G>A and the WT donor were plated onto collagen-coated 96-well plates and once actively proliferating were treated with PBS control, 1010 pfu/mL Pf4, 1 μg/mL LPS, or their combinations. The plate was then placed on an Incucyte incubator (Sartorius Lab Instruments, Goettingen, Germany) at 37 °C and 5% CO2 for time-lapse phase-contrast imaging over a 48 h time span. Conditions were run in quadruplicate, and progression to confluence was quantified by image analysis as we have described before [46 (link)].
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