A The complete setup of two-layer microfluidic chip comprises of external pneumatic membrane valves and pressure source, to actuate on-chip pneumatic membrane valves open or close, and a graphical user interface, written in MATLAB, to provide instructions and aid in automation of experimental workflow. The microfluidic chip is placed in a stage-top incubator and the cells, on-chip, are imaged using fluorescence microscope. B Two-layer microfluidic chip design where flow in sixteen channels (blue) is controlled using eight control lines (red). The inlet channels on the chip are controlled by additional control lines (labeled 9–16) and the outlet is controlled by control port 17. Each flow channel is integrated with cell isolation unit, in green, that has pillar-like traps to physically isolate single cells upon contact. C The pillars, fabricated from PDMS, are separated by a small gap of 4 µm and incorporates a V-shaped cup to efficiently hold individual cells in place. D The control lines, in the top layer, upon orthogonally intersecting the flow lines, in the bottom layer creates several thin pneumatic push-down membrane valves on-chip. The pneumatic membrane valves are actuated open or close through an external pressure source that control the flow of reagents in the channels.” Reproduced under Creative Commons Attribution 4.0 International License from Sinha, N., Yang, H., Janse, D. et al. Microfluidic chip for precise trapping of single cells and temporal analysis of signaling dynamics. Commun Eng 1, 18 (2022).