Metabolic Profiling of Colonic DCs

Colonic DCs were isolated from mice gavaged with P. UF or ΔlspA P. UF1 and orally infected with ΔactA L. m using a Pan-DC Enrichment Kit (StemCell Technologies, Vancouver, Canada). For real-time analysis of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), DCs were analyzed using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) as described.^{52 (link)} Briefly, enriched colonic DCs (3 × 10⁵ cells/well, pooled from 3 mice) were analyzed in non-buffered RPMI medium supplemented with 2.5 μM dextrose, 2 nM glutamine, and 1 μM sodium pyruvate. ECAR and OCR were analyzed in response to 1 μM oligomycin, 1.25 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP), 1 μM rotenone and 1 μM antimycin A.

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Ge Y., Gong M., Zadeh M., Li J., Abbott J.R., Li W., Morel L., Sonon R., Supekar N.T., Azadi P., Wang Y., Jones D.P., Li S, & Mohamadzadeh M. (2019). Regulating colonic dendritic cells by commensal glycosylated large surface layer protein A to sustain gut homeostasis against pathogenic inflammation. Mucosal immunology, 13(1), 34-46.

Publication 2019

Pan-DC Enrichment Kit XFe96 Extracellular Flux Analyzer

Antimycin a Carbonyl cyanide phenylhydrazone Cells Colonic Dextrose Glutamine Mice Oligomycin Oxygen consumption Pyruvate Rotenone Seahorse Sodium Stemcell

Corresponding Organization : University of Florida

Other organizations : University of Georgia, Emory University

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Variable analysis

independent variables

Gavage with P. UF or ΔlspA P. UF1
Oral infection with ΔactA L. m

dependent variables

Extracellular acidification rate (ECAR)
Oxygen consumption rate (OCR)

control variables

Non-buffered RPMI medium supplemented with 2.5 μM dextrose, 2 nM glutamine, and 1 μM sodium pyruvate
Oligomycin (1 μM)
Fluoro-carbonyl cyanide phenylhydrazone (FCCP, 1.25 μM)
Rotenone (1 μM)
Antimycin A (1 μM)

controls

Positive control: Not specified
Negative control: Not specified

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