Right here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. states of Akt, GSK-3, p70S6K, S6, Erk1/2, and mTOR and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the KMT6 Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry. Signal transduction from the extracellular microenvironment to the inner compartments of cells involves the interaction, post-translational modification, and translocation of proteins. Several molecular biology technologies (1C4) have been developed for the quantitative analysis of proteins and their modifications in order to reveal signal dynamics, cross-activations of protein signaling networks, or statistical variations of signals between cells. Predominant are Western blot, time-lapsed fluorescence microscopy, and immunofluorescence assay technologies. For large-scale approaches, however, the standard assays are hampered, although for different reasons. Western blots average millions of cells per data point and provide limited quantitative information. For fluorescence microscopy, long bioengineering processes are required in order to introduce protein labels for each target in a cellular context. In the case of immunofluorescence, the same analytical workflow for the detection of different targets exists (5), but because of the loss of cell integrity during the sample preparation, only one time point per sample can be obtained. The limitation of low sampling rates also holds true for the proximity ligation assay (PLA).1 The PLA technology is a versatile immuno-based detection system for protein interactions, modifications, concentrations, and cellular location (6). The simplest PLA setup for measuring protein concentrations or modifications requires a primary antibody (Ab) that binds its specific target within a fixed cell. A pair of polyclonal secondary Abs conjugated to different oligonucleotide strands is then used to detect the target bound to the primary Ab. In cases where two differently labeled secondary Abs are in close proximity, the oligonucleotide sequences can be complemented, ligated, and amplified by means of rolling circle amplification. Detection of the amplified DNA is achieved through hybridization of a complementary fluorescence probe to the amplified DNA sequence. Positive single PLA events result in a localized DNA polymer with a hydrodynamic diameter of less than 1 m, which can be detected with low numerical aperture optics (6C8). Similar workflows with two primary Abs exist for the detection of protein interactions (7). SNX-5422 Inherent to all currently applied protein assays for cell signaling studies are low integration levels. Workflows for cell cultivation, stimulation, and protein analytics are separated from one another, which leads to low temporal and chemical control over cell samples with the consequence of low comparability between repeats SNX-5422 SNX-5422 or experimental time series. Integrated microfluidic chip technologies can overcome the limitations encountered in large-scale protein analytics. Microfluidics is the science of fluids and their control in micrometer-sized structures (9). Through miniaturization, complex biological workflows can be automated and multiplexed. The advances of microfluidics for cell signaling have been focused mainly on spatial and temporal control over cell microenvironments (10). Chip platforms combining time-lapsed microscopy with automated cell culturing or with fully integrated workflows of immunofluorescence assays (11) are the first steps toward complete analysis systems. But the miniaturization of standard protein assays can also lead to greater precision and throughput, as was shown for Western blots performed in micrometer capillaries (12) and immunoprecipitation assays in microchambers (13). Here, we demonstrate the full implementation of the proximity ligation assay on a microfluidic chip for profiling high-content information of cell signaling pathways. The microfluidic chip is made of multilayered polydimethylsiloxane (PDMS) and combines a perfusion system for cell culturing and stimulation with a multiplexed PLA. About 540 microfluidic PDMS membrane valves operated by 24 electrical solenoid valves were programmed to control the pressure-driven flow of 24 different fluids through the microchannels and chambers of the chip. Cell chambers were arranged in a two-dimensional matrix design in which each axis of the matrix allowed the sampling of a different assay parameter. A cell chamber is able to hold about 100 fibroblast cells. Precise temporal changes of fluids along each column element were used to vary the stimulation times of fibroblast cells with platelet-derived growth factor (PDGF) in a time frame of minutes to hours. Fixation of the cell cultures with paraformaldehyde at different time points after stimulation maintained the.