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

## TL;DR

This paper provides a detailed protocol for studying how changes in cell metabolism affect calcium signaling using flow cytometry and live-cell imaging.

## Contribution

A new protocol is introduced to analyze calcium signaling in response to metabolic changes in a physiologically relevant setting.

## Key findings

- The protocol enables analysis of calcium signaling in a large number of cells.
- It includes steps for cell starvation and stimulation followed by flow cytometry and microscopic analysis.
- The method allows for assessing calcium responses to metabolite stimulation.

## Abstract

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

•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

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 (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.