Measuring Intracelullar Signaling

The immune responses elicited after a cell has been exposed to harmful or toxic stimuli include the activation of different intracellular signaling pathways and 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. An alternative, more streamlined approach is to use multiplexed, automated, quantitative cell-based imaging assays (i.e., high-content screening; HCS) to monitor the intracellular inflammatory responses . In this application example, Raw 264.7 macrophages were challenged with bacterial endotoxin (LPS) and interferon gamma to activate 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 the Thermo Scientific Cellomics ArrayScan VTI HCS Reader and corresponding Thermo Scientific Cellomics HCS Reagents The early targets (MAPK, PKA and transcription factors), were measured after 30 minutes of stimulation, while MnSOD, COX-2 and iNOS were measured at the 24-hour time point. All targets were dose-responsive to the LPS/IFN treatment. The results clearly demonstrate both the power of quantitative cell-based imaging systems for studying signal transduction pathways.

Raw 264.7 cells were stimulated with 500 ng/ml LPS and 100 ng/ml IFN gamma for quantitative image analysis of ten intracellular targets. Early targets (p38, ERK, JNK, PKA, c-Jun, CREB, and NF-kB) were measured after 30 minutes while later targets (MnSOD, COX-2 and iNOS) were measured at 24 hours. (A) Early targets, p38, JNK, ERK, c-Jun, and CREB. (B) Early targets, NF-kB and PKA, and later targets, COX-2, iNOS, and MnSOD. NF-kB has a high background in this cell line, and there is little change after LPS stimulation. However, NF-kB activation may be blocked by pre-treatment with Bay 11-7082 (5 mM) before LPS/IFN challenge. (C) Bar graph, The bar graph represents quantitative data for each target, and is indicated as fold change after normalization to the control (unstimulated). Z’ values are indicated at the top for each target and indicate the robustness of the assay. Error bars represent the standard deviation of each value derived from 16 data points. MnSOD, COX-2 and iNOS were scored using the average cytoplasmic intensity, while the other targets, ERK, p38, c-Jun, NF-kB, CREB, PKA, and JNK were quantitated using the average nuclear intensity or difference between nucleus and cytoplasm.