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. 2022 Feb 18;3(1):101186.
doi: 10.1016/j.xpro.2022.101186. eCollection 2022 Mar 18.

Flow-cytometry-based protocol to analyze respiratory chain function in mouse microglia

Daniel Erny  1   2 , Nikolaos Dokalis  1   3 , Charlotte Mezö  1   3 , Omar Mossad  1   3 , Thomas Blank  1 , Marco Prinz  1   4   5
Affiliations

Flow-cytometry-based protocol to analyze respiratory chain function in mouse microglia

Daniel Erny et al. STAR Protoc. .

Abstract

Most of the protocols to analyze metabolic features of cell populations from different tissues rely on in vitro cell culture conditions. Here, we present a flow-cytometry-based protocol for measuring the respiratory chain function in permeabilized mouse microglia ex vivo. We describe microglial cell isolation, followed by analyzing complex I and II using flow cytometry. This optimized protocol requires a low input of permeabilized cells and can be applied to other ex vivo isolated cells or cells derived from cell cultures. For complete details on the use and execution of this protocol, please refer to Erny et al. (2021).

Keywords: Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Metabolism; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Gating strategy for flow cytometric analysis of single, live, CD11b+CD45low MitoTracker Green+ microglia The last dot plot depicts the back gating of CD11b+CD45low cells. Representative dot plots are shown. FSC: forward scatter. SSC: side scatter.
Figure 2
Figure 2
Examination of complex I function (A) Representative cytometry graphs of the mitochondrial membrane potential Δψm (tetramethylrhodamine, TMRM) which was manipulated by (B) malate & pyruvate (Mal/Pyr) and subsequently by (C) carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) in CD11b+CD45low microglia.
Figure 3
Figure 3
Examination of complex II function (A) Representative cytometry graphs of the mitochondrial membrane potential Δψm which was manipulated by (B) rotenone (Rot) and subsequently by (C) succinate (Succ.) and (D) antimycin A1 (AA1) in CD11b+CD45low microglia.
Figure 4
Figure 4
Examination of complex II function (A) Representative cytometry graphs of the mitochondrial membrane potential Δψm which was manipulated by rotenone (Rot) and subsequently by (B) oxaloacetate (OAA) and succinate (Succ.) in CD11b+CD45low microglia.
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