Protocol to assess calcium signaling in response to metabolic alterations by flow cytometry and live-cell imaging

Calcium (Ca²⁺) signaling is essential to cellular processes, such as signal transduction and migration, and dysregulation of Ca²⁺ homeostasis can impact cellular function. Here, we present a protocol to analyze Ca²⁺ signaling by its release into the cytosol in response to and after metabolic stimulation. We describe the steps for cell starvation from excessive nutrients and stimulation, followed by flow cytometry-based and microscopic analysis of calcium signaling. This protocol enables analysis of a large number of cells in a physiologically relevant microenvironment. For complete details on the use and execution of this protocol, please refer to Schlautmann et al.¹.