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. 2022 Jun 16;9:923502. doi: 10.3389/fmed.2022.923502

FIGURE 2.

FIGURE 2

Beta-hydroxybutyrate shifts human T-cell metabolism toward oxidative phosphorylation enabling higher mROS production. Human peripheral blood mononuclear cells (PBMC) were cultivated for 5 days in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM D/L-beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD8+ T cells were isolated via magnetic cell separation. (A–D) Oxygen consumption rate [OCR] (A), basal (B), maximum (C) and spare respiratory capacity (D) were measured using a Seahorse HS mini Analyzer, n = 5 individual patient samples, each performed in 2–3 technical replicates. (E) Mitochondrial mass determined via MitoTracker green, indicated by MFI FITC in human CD8+ T cells, n = 12 individual patient samples. Histogram depicting exemplary change of MitoTracker green. (F) Quantification of mitochondrial superoxide production using MitoSOX, displayed as MFI PE in human CD8+ T cells, n = 11 individual patient samples. Histogram depicting exemplary change of MitoSOX fluorescence. *p < 0.05, **p < 0.01.