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We propose a microfluidic strategy for investigating cellular dynamics in single cellular and intercellular scale by real-time addressing flow,which takes advantage of the flexible hydrodynamic gating valve and hydrodynamic focusing,enabling stimulation of adherent cells with pre-addressed micro-flow of agonist.In this approach,theory was validated by both numerical simulations and flow visualization experiments.A computer-aided pressure control system was constructed to realize the rapid real-time addressing and stimulating of pre-addressed micro-flow with high precision,high temporal(>12 ms)and spatial resolutions(16.3±2.4 μm).The intracellular and intercellular calcium signals were successfully induced by millisecond to second localized stimulation of the target single cells.These results reveal that the proposed microfluidic strategy provides a versatile means for probing cellular dynamics,which can also be potentially useful in c.hemical biology,cell biology and pharmacology.