Chapter 29
A Flow Cytometry-Based Protocol to Measure Lymphocyte
Viability Upon Metabolic Stress
Se ́bastien Denanglaire, Tiphe`ne Pirnay, Oberdan Leo,
and Fabienne Andris
Abstract
Distinct lymphocyte subpopulations display discrete metabolic profiles and are differently affected by
metabolic resource variations, making the analysis of lymphocyte survival in a complex tissue in response
to metabolic stress highly challenging. Here we describe a flow cytometry-based method allowing simulta-
neous cell identification and viable cell counting in mixed lymphocyte populations without extensive cell
subset purification procedures. The example provided herein illustrates the role of AMPK in T lymphocyte
survival in response to the mitochondrial poison oligomycin.
Key wordsFlow cytometry, Polystyrene beads, Lymphocytes, Cell counting, Antibodies, Metabolic
stress1 Introduction
AMP-activated protein kinase (AMPK) is a critical energy sensor
adapting cellular metabolism in response to environmental varia-
tions, thus playing a crucial role in maintaining cellular energy
metabolism homeostasis [1, 2]. AMPK is switched on by cellular
stresses that either increase ATP consumption (i.e., muscle contrac-
tion) or by stresses that interfere with ATP production (i.e., hyp-
oxia, glucose deprivation, or mitochondrial poisons) [3, 4].
Differentiation of immune cells in effector or long-term mem-
ory cells is tightly associated with changes in energy metabolic
activity that allow the cells to balance requirements for energy or
molecular biosynthesis [5–8]. Moreover, during immune
responses, activated lymphocytes encounter distinct metabolic
environments with altered availability of nutrients and must adapt
to survive [9]. This is particularly relevant in antitumor immunityDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_29,©Springer Science+Business Media, LLC 2018
Se ́bastien Denanglaire and Tiphe`ne Pirnay are Co-first authors
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