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P2058
Assessment of the Immune Response Using High-content Screening Cell-based Imaging & Antibody Arrays
Presenter Brian Webb, Thermo Fisher Scientific
Additional Authors: Kay Opperman, Eric Hommema, and Richik N. Ghosh
The immune responses elicited after a cell has been exposed to harmful or toxic stimuli include the activation of different intracellular signaling pathways, leading to the release of cytokines. The signaling pathway and severity of the response is both cell type-specific and stimulus-dependent. The traditional approach to examining multiple intracellular targets and subsequent cytokine production requires numerous assays, multiple extract preparations, increased sample size and a significant amount of time. We present here a more streamlined approach for quantifying the activation of key intracellular signaling pathways and correlating it with cytokine release. We combined multiplexed automated, quantitative cell-based imaging assays (i.e., high-content screening; HCS) with antibody arrays to monitor and correlate both the intracellular and extracellular inflammatory responses. Raw 264.7 macrophages were challenged with bacterial endotoxin (LPS) and interferon gamma. This potent combination results in signal transduction through the TLR and IFN receptors, yielding robust activation of many target proteins and cytokines. A total of 10 key targets, including p38, ERK, JNK, c-Jun, NF-kB, CREB, PKA, COX-2, iNOS and MnSOD, were quantitatively measured using a Thermo Scientific Cellomics® ArrayScan® HCS Reader and Cellomics HCS Reagent Kits for the above targets. The early-response targets, including MAPK, PKA, and transcription factor activation, were measured after 30 minutes of stimulation, while COX-2 and iNOS were measured at the 24-hour time point. All targets were dose-responsive to the LPS/IFN treatment. To further demonstrate the usefulness of this approach for compound library screening, cells were then pre-treated with commercially available anti-inflammatory compounds to measure inhibition of the immune response. Dose response curves were generated varying drug concentration for drug IC50 values. Using a Thermo Scientific Mouse ExcelArray™ Inflammation Array, alterations in the target proteins were then correlated with different cytokine responses. Using these two approaches, compound target specificity was determined and further correlated to the inflammatory response. Compounds were then grouped using IC50 values, target inhibition and cytokine response. Results from this study demonstrate the power of quantitative cell-based imaging systems in the study of signal transduction pathways and the enhanced value of multiplexing different compatible technologies.