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

Calcium (Ca 2+ ) signaling is essential to cellular processes, such as signal transduction and migration, and dysregulation of Ca 2+ homeostasis can impact cellular function. Here, we present a protocol to analyze Ca 2+ 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 • Steps for analyzing calcium signaling by flow cytometry and live-cell imaging • Detailed instructions on data acquisition and analysis of calcium signaling • Guidance for determining calcium response to metabolite stimulation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Calcium (Ca 2+ ) signaling is essential to cellular processes, such as signal transduction and migration, and dysregulation of Ca 2+ homeostasis can impact cellular function. Here, we present a protocol to analyze Ca 2+ 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.