Cell Signaling Technology (CST) offers a growing line of cell based assay reagents and kits designed with the drug discovery investigator in mind. Whether your research calls for a primary cell based screen, or you are performing secondary cell based assays, CST offers an extensive line of phosphorylation-specific and total antibodies, substrates and assay kits to meet your needs. Moreover, CST has validated over 500 activation state-specific (e.g., phosphorylation-specific) and total protein antibodies for immunofluorescence (IF) applications such as automated cell based assays. For the characterization of select pathways of interest, CST has also developed Multi-Target IF Kits that contain panels of antibodies pre-optimized for use in whole cell assays.

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CST Antibodies in High Content Screening

The Acumen® eX3 (*TTP LabTech, www.ttplabtech.com/products/acumen/) is a laser scanning fluorescence microplate cytometer used for high content screening. CST’s IF-validated activation state-specific and control antibodies are routinely used in-house on this high content platform both for single pathway profiling (example A) and for systems biology profiling (example B).

Example A. Single Pathway Profiling: A MAPK pathway inhibitor was tested in HeLa cells with anisomycin stimulation. Three downstream endpoints (phospho-CREB, phospho-Akt, and phospho-HSP27) were chosen to assess the efficacy of the inhibitor. Phospho-Rb and cleaved caspase-3 were also used to examine the effect of the inhibitor on proliferation and apoptosis, respectively. Cells were grown and treated in 96-well plates and labeled with CST antibodies. Plates were analyzed in-house on an Acumen® eX3 high content platform.

With and without Inhibitors MAPK pathway inhibitor

The MAPK inhibitor inhibited cellular signaling as evidenced by the decrease in phospho-Akt and phospho-HSP27 levels (see IC50 values and graphs above), while not increasing toxicity (see minimal change in phospho-Rb and cleaved caspase-3 levels above).

Example B. Systems Biology Profiling: Antibody array data from the Bcr/Abl-positive K562 and Bcr/Abl-negative SEM cell lines treated with dasatinib and imatinib. Cells were grown and treated in 96-well plates and labeled with a panel of 96 CST antibodies. Plates were analyzed in-house on an Acumen® eX3 high content platform and a high throughput flow cytometer.

Cell-based Assay Fluorescence

Responses were reported as either a decrease (red) or increase (green) in fluorescence intensity compared to vehicle. Both drugs inhibited Bcr/Abl-specific signaling pathways in K562 but not SEM cells.

15 inhibited targets - imatinib 15 inhibited targets - dasatinib

Responses of the 96 endpoints were sorted according to percent inhibition as compared to vehicle. The top 15 inhibited targets are represented in these graphs (imatinib-top, dasatinib-bottom). The magnitude of the response and the targets affected were slightly different between the two compounds.

*Data may be generated with methods/devices/compositions requiring a license to relevant third party intellectual property rights.