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. 2021 Mar 26;2(2):100401.
doi: 10.1016/j.xpro.2021.100401. eCollection 2021 Jun 18.

Metabolic analysis of mouse bone-marrow-derived dendritic cells using an extracellular flux analyzer

Kazuhito Gotoh  1 , Yurie Takata  1 , Yuya Nakashima  1 , Soichi Mizuguchi  1 , Keishi Komori  1 , Dongchon Kang  1
Affiliations

Metabolic analysis of mouse bone-marrow-derived dendritic cells using an extracellular flux analyzer

Kazuhito Gotoh et al. STAR Protoc. .

Abstract

Dendritic cell (DC) maturation induced by Toll-like receptor (TLR) agonists requires the activation of downstream metabolic changes. Here, we provide a detailed protocol to measure glycolysis, mitochondrial respiration, and fatty acid oxidation in mouse bone-marrow-derived DCs with the Seahorse XF24 extracellular flux (XF) analyzer. XF analysis with the Seahorse bioanalyzer has become a standard method to measure bioenergetic functions in cells, and this protocol can be adapted to other immune cells. For complete information on using this protocol, please refer to Gotoh et al. (2018).

Keywords: Cell biology; Cell culture; Cell isolation; Cell-based assays; Immunology; Metabolism.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Preparation of palmitate (PA) + bovine serum albumin (BSA) conjugate
Figure 2
Figure 2
Photographs of XFe sensor cartridge and XF24 extracellular flux analyzer
Figure 3
Figure 3
Management of the assay protocol (A) Open software and select a protocol. (B) Select Blank and sample wells. (C) Design a protocol. (D) Check a protocol. (E) Load a calibrant plate with compounds. (F) Finish this assay.
Figure 4
Figure 4
Metabolic change by TLR stimulation Real-time changes in the ECAR and OCR of BMDCs treated with LPS (A,B), CpG (C, D), and IMQ (E, F).
Figure 5
Figure 5
ECAR and OCR of immature and mature BMDCs BMDCs (2 × 105 cells/well) stimulated without (immature) or with (mature) LPS (100 ng/mL) for 12 h were seeded in an XF-24 extracellular flux analyzer. The real-time OCR and ECAR were measured during the sequential treatments with a mitochondrial stress test (A: oligomycin, FCCP, antimycin-A/rotenone) and glycolysis stress test (B: Glucose, oligomycin, 2-Deoxy-D-glucose (2DG)).
Figure 6
Figure 6
OCR of BMDCs for FAO assay Wild-type DCs (2 × 105 cells/well) pretreated with PA+BSA (A) and Etomoxir (B) for 30 min were seeded in an XF-24 extracellular flux analyzer. The real-time OCR was determined during the sequential treatments with a mitochondrial stress test (A: oligomycin, FCCP, antimycin-A/rotenone).
Figure 7
Figure 7
Analysis of XF assay (A) Open software and select OCR or ECAR. (B) Normalization of the assay. (C) Export Excel or Prism. (D) Printscreen of XF Mito Stress Assay.
Figure 8
Figure 8
Statistical analysis of mitochondrial stress test and glycolytic stress test A schematic of a mitochondrial stress test (A) and glycolytic stress test (B) using the extracellular flux analyzer.
Figure 9
Figure 9
Photograph of bone marrow pellets Bone marrow pellet before RBC lysis (A), after RBC lysis (B).
See this image and copyright information in PMC

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