Proximity Ligation Assay for High-content Profiling of Cell Signaling Pathways on a Microfluidic Chip

Abstract

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 (1-4) 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, timelapsed 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 in situ 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

In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip

“…The mPLA on chip increases throughput and allows parallel monitoring of different targets within the same cells compared with geometric multiplexing of the PLA in separate microchambers for cell culturing (41,42). The advantages of the mPLA were exploited for the characterization of the mTORC1 during adipogenesis.…”

“…S5, and all of the used oligonucleotide sequences are given in Table S2. The on-chip PLA is described elsewhere (41). In short, hASCs were fixed after 14 d of culturing with 4% (wt/vol) paraformaldehyde in PBS for 12 min.…”

Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices

“…In another approach, Blazek and colleagues 106 used proximity ligation assays (PLAs) 107 to measure the abundance of a single protein in a few cells. Unlike DEAL, PLA relies on the binding of two oligonucleotide-tagged antibodies in proximity to generate, with a helper bridge oligonucleotide, a DNA reporter with low background noise 108 .…”

Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation

“…Image sets included nuclei (21HE filter), cytoskeleton (38HE), and RCA signal (43HE); integration times were adjusted manually and kept constant for the entire experiments. Single cell dot counts were acquired by image analysis (Blazek et al 2013) with the MATLAB Image Processing Toolbox (Mathworks).…”