# Protocol for measuring cellular energetics through noncanonical amino acid tagging in human peripheral blood and murine tissue immune cells

## TL;DR

This paper introduces a new protocol to study immune cell metabolism using noncanonical amino acid tagging in human and mouse immune cells.

## Contribution

A novel flow cytometry-based protocol (CENCAT) for profiling immunometabolism in low-yield immune cell samples.

## Key findings

- A detailed protocol for measuring glucose and mitochondrial dependence in immune cells.
- Steps for βES incorporation and fluorescent tagging of nascent proteins via click chemistry are described.
- The protocol enables metabolic profiling of human PBMCs and murine tissues.

## Abstract

Cellular metabolism dictates immune cell function, yet we lack tools to functionally profile immunometabolism in low-yield, complex samples. We present a flow cytometry-based protocol for measuring cellular energetics through noncanonical amino acid tagging (CENCAT) in human peripheral blood and murine tissue immune cells. We describe steps for sample preparation, metabolic inhibition, protein synthesis analysis using click chemistry, immunophenotyping, and calculation of metabolic dependencies.

For complete details on the use and execution of this protocol, please refer to Vrieling et al.1

•Instructions for preparing human PBMCs and murine tissues for metabolic profiling•Protocol for βES incorporation in nascent proteins under metabolic inhibition•Step-by-step guide to fluorescent tagging of nascent proteins via click chemistry•Procedure for calculating relative cellular glucose and mitochondrial dependence

Instructions for preparing human PBMCs and murine tissues for metabolic profiling

Protocol for βES incorporation in nascent proteins under metabolic inhibition

Step-by-step guide to fluorescent tagging of nascent proteins via click chemistry

Procedure for calculating relative cellular glucose and mitochondrial dependence

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Cellular metabolism dictates immune cell function, yet we lack tools to functionally profile immunometabolism in low-yield, complex samples. We present a flow cytometry-based protocol for measuring cellular energetics through noncanonical amino acid tagging (CENCAT) in human peripheral blood and murine tissue immune cells. We describe steps for sample preparation, metabolic inhibition, protein synthesis analysis using click chemistry, immunophenotyping, and calculation of metabolic dependencies.