By W. Frank An, Nicola J. Tolliday (auth.), Paul A. Clemons, Nicola J. Tolliday, Bridget K. Wagner (eds.)
As using high-throughput screening expands and creates extra curiosity within the educational group, the necessity for particular reference fabrics turns into ever extra urgent. Cell-Based Assays for High-Throughput Screening: equipment and Protocols goals to fill an incredible a part of this desire by means of offering an simply obtainable reference quantity for cell-based phenotypic screening. best researchers within the box give a contribution state of the art equipment with actionable protocols protecting 4 significant parts of analysis: version organic structures, screening modalities and assay platforms, detection applied sciences, and methods to info research. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, each one bankruptcy encompasses a short advent to the topic, lists of priceless fabrics and reagents, step by step laboratory protocols, and a Notes part detailing tips about troubleshooting and averting recognized pitfalls.
Cutting-edge and easy-to-use, Cell-Based Assays for High-Throughput Screening: tools and Protocols provides an summary of proper techniques, allowing the direct software of latest how to new discoveries whereas additionally inspiring researchers to technique their screening tasks in a conceptually modular style, improving the ability to find via new mixtures of present approaches.
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Additional info for Cell-Based Assays for High-Throughput Screening: Methods and Protocols
Cells were stained with the phosphorylation-specific antibodies against CREB as described above. As shown in Fig. 5, the nuclear accumulation of phosphorylated CREB shows a strong dependence on the concentration of TPA. 7. The plateau is reached at 250 nM of TPA and the AC50 is 15nM. TPA-induced nuclear accumulation of phosphorylated CREB is mediated by active MEK as shown in Fig. 6. cn ←点击进入 Cell-Based Assays to Probe the ERK MAP Kinase Pathway 39 Fig. 5. TPA inducing phosphorylation of Ser133 of CREB in a dose-dependent manner.
Spray the bench surface in front of the liquid handler with 95% ethanol and wipe down. Remove the lids from the sterile 96-well plates by beginning with the row farthest from the operator, and working towards the operator; this minimizes hand movement over open plates, a potential source of contamination. 20. Recall that the control block contains reagent in columns 1 and 12 only, whereas the same columns in the compound storage plates are empty. 0 0 5 10 15 20 25 Time (hr) 30 35 40 Fig. 1. E. coli growth over time in the presence of 5% DMSO.
Incubate for 20 min. 10. Remove antibody solution and wash three times with 100 μL PBS. 11. Seal and store at 4°C in fresh 100 μL PBS until ready to scan. 4. Image Acquisition 1. Software settings (“Nuclear Traffic Module” of INCell Analyzer 3000 software). 00 • Fill Holes: Y • Erosion: 0 2. Plates were imaged by sequential scanning using the red and blue channel. 3. To acquire the first image, samples were excited with the 647 nm laser line and images were recorded with the 695– 55nm emission filter (red channel).