Calcium (Ca2+) is a universal second messenger in eukaryotic cells, and extracellular regulated protein kinases (ERK) are the core component of the mitogen-activated protein kinase (MAPK) signaling cascade. Both are involved in numerous physiological and pathological processes, such as organogenesis, tumorigenesis, proliferation, migration and apoptosis. Over the past decade, it has been found that calcium signaling can regulate the ERK activity through multiple mechanisms, and conversely, ERK signaling transduction can also affect the triggering and intensity of calcium signaling. However, there are few reports on how to perform real-time synchronous detection of these two signals. Here we described a method for dynamically and synchronously recording calcium signals and ERK activity in living cells, utilizing stable expression of multiple genetically-encoded probes and multi-channel synchronous detection technology using confocal microscopy. The protocol can be useful to address the spatiotemporal encoding dynamic mechanism of calcium signaling and ERK activity in single or multiple cells, and to reveal the interaction and causal characteristics of these two signals.
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