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.
Enspire multimode plate reader instrument
Manufactured by PerkinElmer
Sourced in United States
The EnSpire® Multimode Plate Reader is a versatile instrument designed for various laboratory applications. It offers a range of detection modes, including absorbance, fluorescence, and luminescence, allowing researchers to perform a variety of assays and experiments. The EnSpire® Plate Reader is capable of handling microplates of different sizes and formats, providing flexibility in experimental design.
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Lab products found in correlation
3 protocols using enspire multimode plate reader instrument
1
Quantitative p53 Protein Analysis
2
Crystal Violet Cell Proliferation Assay
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The cell proliferation was evaluated by a crystal violet assay. The cells were seeded in a 6-well plate (35 × 103 cells/well), and an assay was performed 48, 72, 96, 120, and 144 h after seeding. At the end of each established time point, the cells were washed with 1× Phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in 1× PBS for 20 min at room temperature. Then, the cells were incubated with a solution containing 0.5% crystal violet (Sigma-Aldrich, Steinheim, Germany) in 20% methanol for 15 min. After careful washing with H2O, the dye was extracted with a 0.1 M sodium citrate solution in 50% ethanol (pH 4.2) and quantified at 540 nm with an EnSpire® Multimode Plate Reader instrument (PerkinElmer, Waltham, MA, USA).
3
Caspase-3/7 Activity Measurement in Cells
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A Caspase-Glo® 3/7 assay kit (Promega, Madison, WI, USA) was used to evaluate the activity of caspases 3/7. Briefly, the cells were seeded in a 96-well plate (30 × 103 cells/well). After 72 h, the activity of caspase 3/7 was determined in accordance with the manufacturer’s protocol. In the MG132 treatment and siRNA experiments, the caspase 3/7 activity was determined at an established time point (Figure S6 ). The luminescent signal was measured by the EnSpire® Multimode Plate Reader instrument (PerkinElmer, Waltham, MA, USA) and normalized for DNA content, determined by a CellToxTM Green Cytotoxicity Assay kit (Promega, Madison, WI, USA). The fluorescent signal was measured with EnSpire® Multimode Plate Reader instruments (PerkinElmer, Waltham, MA, USA).
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