The levels of p53 protein were quantitatively analysed using the Alphascreen SureFire Total p53 Assay Kit (TGR Biosciences/Perkin Elmer #TGRT53). Briefly, A549 cells (40,000 cells/well) were reverse transfected with 40 nM siRNA (final reaction volume 0.5 mL) in Nunc 24-well cell culture plates for 72 hours, refreshing media at 24 hours’ post transfection. Just prior to harvest, to normalise p53 signal, plates were imaged on the IncuCyte S3 Live-Cell Analysis Imaging System (Essen Biosciences) using the scan-on-demand feature (2015A software release), calculating the Phase Object Confluence (Percent) for each well. Media was then carefully removed, and the cell monolayers lysed with 30 μL 1x Alphascreen Lysis Buffer for 2-3 minutes on an orbital rocker, prior to transfer of lysates to −20°C. Lysates (4 μL/well in Perkin Elmer 384 well proxiplates, #6008280) were then assayed in as per the manufacturers’ instructions. Plates were then read using the Perkin Elmer EnSpire Multimode Plate Reader instrument using the instrument pre-defined Alphascreen settings and the data normalised to the cell confluence readings, then normalised to the non-targeting siRNA treated wells.
Incucyte s3 live cell analysis imaging system
Manufactured by Sartorius
Sourced in United States
The IncuCyte S3 Live-Cell Analysis Imaging System is a real-time, automated cell imaging platform that allows for continuous monitoring and quantitative analysis of live cells in their native environment. The system captures high-quality images at regular intervals, enabling researchers to track cell behavior, morphology, and proliferation over time.
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Lab products found in correlation
2 protocols using incucyte s3 live cell analysis imaging system
1
Quantitative p53 Protein Analysis
2
Real-time monitoring of PLGA NP-cell interactions
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In order to investigate the interaction between PLGA NPs displaying a protein corona of different characteristics and HepG2 cells an IncuCyte®S3 Live-Cell Analysis Imaging System (Essen Bioscience, Inc., Michigan, USA) was used. Therefore, 1 × 105 cells/well were seeded into a collagen coated 24-well plate and cultivated under serum-containing conditions as described above. After four days, the medium was replaced by 500 µL serum-free medium containing Lumogen® Red-loaded PLGA NPs in a concentration corresponding to 0.2 nM Lumogen® Red. Furthermore, unformulated Lumogen® Red dissolved in serum-free medium with addition of 1% DMSO and serum-free medium without NPs were applied onto the cells as control. Subsequently, the cell interaction was monitored over a time period of one day taking nine images of each well every hour. Image channel red (excitation: 565–605 nm/emission: 625–705 nm) was used to determine Lumogen® Red. Data evaluation was performed by calculation of the total red object area using the system software.
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