Prostate cancer cells with constitutive DsRed expression were plated into 24-well black-walled, glass-bottom plates at 10% confluence. The cells were treated as with previous experiments in quadruplicate with treatments replenished at day 3. At day 0, 3 and 6, each well was imaged using a BioTek Lionheart FX automated microscope (BioTek, Winooski, VT) with a 4x/0.13NA objective (Olympus, Tokyo, Japan) and an RFP filter cube (531 nm/593 nm) paired with a 523 nm high power LED. The images were analyzed for confluency using the Gen5 Image Prime software package (BioTek, Winooski, VT).
Lionheart fx automated
Manufactured by Agilent Technologies
Sourced in United States
The Lionheart FX is an automated microscope system designed for live-cell imaging and analysis. It features advanced optics, environmental controls, and imaging capabilities to support a range of cell-based assays and applications.
Automatically generated - may contain errors
6 protocols using lionheart fx automated
1
Monitoring Prostate Cancer Cell Growth
2
Automated Live Cell Imaging Workflow
3
Cell Migration and Invasion Assay Protocol
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Cell migration assays were performed using a 24-well 8.0 µm polycarbonate transwell chamber (Corning, Inc., Corning, NY, USA), and Matrigel-precoated transwell inserts (BD Biosciences, San Jose, CA, USA) were used for the invasion assay.
For the migration assay, A2780 and A2780-ADR cells (5 × 105 cells/mL) were suspended in high-glucose, serum-free DMEM; the upper chamber was filled with 100 µL cells, whereas the bottom chamber was filled with 600 µL DMEM containing 10% FBS with etravirine (0, 5, and 10 µM), followed by incubation at 37 °C and 5% CO2 for 24 h. After the incubation period, the insert was carefully removed. Cells on the lower side of the insert membrane were fixed with 4% paraformaldehyde for 10 min, followed by staining with 0.5% crystal violet in 25% methanol for 30 min at room temperature. The insert was washed twice with PBS for several seconds to remove excess dye. Cells in the upper part of the insert were removed by gently swiping with a cotton swab. Finally, the migrating cells were counted using a Lion Heart FX automated microscope (Biotek, Winooski, VT, USA). Experiments were performed in triplicate for each group.
For the invasion assay, all steps were the same as those described above, but the incubation time was 36 h.
For the migration assay, A2780 and A2780-ADR cells (5 × 105 cells/mL) were suspended in high-glucose, serum-free DMEM; the upper chamber was filled with 100 µL cells, whereas the bottom chamber was filled with 600 µL DMEM containing 10% FBS with etravirine (0, 5, and 10 µM), followed by incubation at 37 °C and 5% CO2 for 24 h. After the incubation period, the insert was carefully removed. Cells on the lower side of the insert membrane were fixed with 4% paraformaldehyde for 10 min, followed by staining with 0.5% crystal violet in 25% methanol for 30 min at room temperature. The insert was washed twice with PBS for several seconds to remove excess dye. Cells in the upper part of the insert were removed by gently swiping with a cotton swab. Finally, the migrating cells were counted using a Lion Heart FX automated microscope (Biotek, Winooski, VT, USA). Experiments were performed in triplicate for each group.
For the invasion assay, all steps were the same as those described above, but the incubation time was 36 h.
4
Live-cell Imaging of Peptide-induced Cytotoxicity
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Prior to treatment, 10,000 MCF7-G11-TR5 cells were plated in a 96 well plate 18 h. Following B18L treatment, 10x live-kinetic Brightfield images were captured at 30 s intervals for 45 min using the Lionheart FX automated microscope’s experiment mode feature (Biotek, Winooski, VT, USA). Before treatment with B18L, images were taken as time 0. For live/dead cell stain, cells were pre-stained with Cell viability imaging kit (blue/green) for 1 h prior to treatment with peptide. Cells were treated with peptides (IC50 concentration) and 10x live kinetic images were taken at 5-min intervals for 45 min. The area of the cells, membrane blebs and dead cell intensity were quantified using ImageJ 1.52a [57 (link)].
5
Automated Live Cell Imaging Workflow
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Cells were cultured in 6-well tissue culture dishes. After 48 hours, cells were fed and live cell imaging was performed using a Lionheart FX automated live cell imager (BioTek). Beacons were set for each cell type using Gen5 software. The LionHeart FX captured 3 × 3 20X montages at 5 min intervals for 21–28 hours for each beacon. Using BioTek Gen5 (version 3.02) software, montages were stitched together and movies were created.
6
Viability Assessment of 3D Printed Cells
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During deposition the cell-laden filament through dispensing nozzle, the cell experiences shear stress, which could be potentially harmful to the cell. Thereby, the cell viability and cytotoxicity is conducted using LIVE/DEAD assay after the printing and at the different time period. ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Thermofisher, Waltham, MA, USA) was used following the manufacturers protocol. The filament with the cells was imaged using Lionheart FX automated live cell imager (Biotek, Winooski, VT, USA). The z-stack images are captured using 50 μm layer thickness. The protocol is defined accordingly and beacon (n = 5) is selected randomly. Laser power and other detector parameters are kept constant throughout the imaging of the different beacons. The percentage of viability is determined using the following equation:
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