By W. Frank An, Nicola J. Tolliday (auth.), Paul A. Clemons, Nicola J. Tolliday, Bridget K. Wagner (eds.)

ISBN-10: 1603275444

ISBN-13: 9781603275446

As using high-throughput screening expands and creates extra curiosity within the educational group, the necessity for specified reference fabrics turns into ever extra urgent. Cell-Based Assays for High-Throughput Screening: tools and Protocols goals to fill a major a part of this want by way of supplying an simply obtainable reference quantity for cell-based phenotypic screening. best researchers within the box give a contribution state of the art tools with actionable protocols protecting 4 significant parts of analysis: version organic structures, screening modalities and assay platforms, detection applied sciences, and techniques to facts research. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, every one bankruptcy contains a short creation to the topic, lists of valuable fabrics and reagents, step by step laboratory protocols, and a Notes part detailing tips about troubleshooting and fending off identified pitfalls.

Cutting-edge and easy-to-use, Cell-Based Assays for High-Throughput Screening: equipment and Protocols provides an outline of appropriate ways, allowing the direct software of latest how you can new discoveries whereas additionally inspiring researchers to strategy their screening initiatives in a conceptually modular style, bettering the facility to find via new mixtures of current approaches.

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4). 4. To initiate activation of the pathway, the medium was removed, and 100 μL of EGM-2 medium (not supplemented) containing 1 μM TPA was added. 5. After 30 min incubation at standard conditions, cells were fixed by adding 35 μL of 16% paraformaldehyde (see Note 3). Fig. 4 shows images of HUVECs treated with (Fig. 4B, right panel) or without TPA (Fig. 4B, left panel). Images were taken with the INCell Analyzer 3000. 6. Cells were stained with the phosphorylation-specific antibodies against CREB as described above.

HUVECs were seeded at 10,000 cells per well (96-well Packard view plate, as described above) and incubated for 96 h in EGM2 medium at standard conditions. 2. A 96 h incubation will deplete growth factors from the medium and therefore greatly reduce the baseline amount of phosphorylated CREB (see Note 5). 3. As was done for the ERK1/2 assay, 1 μM TPA for 30min was used as an activator in the present assay (Fig. 4). 4. To initiate activation of the pathway, the medium was removed, and 100 μL of EGM-2 medium (not supplemented) containing 1 μM TPA was added.

3. In general, we find that it is not necessary to remove the medium or wash the cells before adding the fixative. 4. For most antibodies, 20-min incubation at room temperature is sufficient. 5. We find that the 96-h starvation period is an easy and reproducible way to induce quiescence in HUVECs. Acknowledgments This work was supported by a MLSCN grant #1U54 H600391401 to James E. M. E. Rothman for his support and Dr Martin Wiedmann for his input and constant suggestions during the progress of the work.

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Cell-Based Assays for High-Throughput Screening: Methods and Protocols by W. Frank An, Nicola J. Tolliday (auth.), Paul A. Clemons, Nicola J. Tolliday, Bridget K. Wagner (eds.)


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