Table 1.
Description of subjects.
Fig 1.
Schematic of oxygen consumption trace from extracellular flux analysis.
Oligomycin, ATP synthase inhibitor; 1 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP), ionophore which shuttles hydrogen ions; rotenone and antimycin A, complex I and III inhibitor respectively, are added to cells sequentially to assess mitochondrial respiration.
Table 2.
Summary of SeahHORse Explorer (SHORE) program development.
Fig 2.
Optimization of mitochondrial perturbation chemicals including (A) oligomycin, (B) FCCP, (C) antimycin A, and (D) rotenone for measurement of mitochondrial function in human immune cells. Data is represented as the % of the basal OCR after addition of the indicated drug. N = 3. For all figures, values in the same panel with the same letter are statistically indistinguishable, values with different letters are statistically different as analyzed by 1-way ANOVA.
Fig 3.
Titration of cell number per well for bioenergetics measurements in human PBMCs and CD4+ T cells.
(A) Mitochondrial stress test seahorse profiles. (B). The relationship between the maximal respiration vs cell number is linear. (C) Ratio of the maximal respiration to the basal respiration is plotted against cell number. Resting PBMCs (N = 4), Activated PBMCs (N = 3), Resting T cells (N = 2), Activated T cells (N = 3).
Fig 4.
Technical considerations for performing flux analysis with primary human immune cells.
(A) CD4 T cells were activated using anti-CD3/CD28 Dynabeads or a plate bound anti-CD3 and soluble anti-CD28 method for 40hrs. (N = 3) (B) Before running flux analysis, PBMCs were kept at room temperature (RT) or on ice during samples preparation (N = 3). (C) CD4+ T cells, isolated from PBMCs, were immediately activated for 40hrs and then frozen (Activated→Frozen) or CD4+ T cells, isolated from PBMCs, were first frozen, followed by T cell activation for 40hrs, then immediate flux analysis (Frozen→Activated) (N = 2). (D) Mitochondrial stress test profile from paired fresh (never frozen) PBMCs and PBMCs frozen for 6 months (N = 2). The mean centered basal respiration values of (E) resting PBMCs (N = 2) fresh or frozen for various amounts of time before flux analysis.
Fig 5.
Bioenergetics Activation time course of CD4+ T cells.
(A) OCR, (B) ECAR, (C) Basal Respiration, (D) Maximal Respiration, (E) Spare Respiratory Capacity, (F) OCR to ECAR ratio, and (G) secreted IL-6 of CD4+ T cells stimulated with anti-CD3/CD28 for indicated time. N = 3.
Fig 6.
Mitochondrial respiration in PBMCs from T1D and T2D subjects.
(A) OCR Mito Stress test Seahorse profiles and area under the curve analysis (B) for resting PBMCs from subjects with T2D (N = 11) or T1D (N = 6). (C) Metabolic parameters and (D) OCR:ECAR at basal and maximal respiration derived using the Seahorse analysis program. Unpaired two-tailed student’s t test for T2D vs T1D.
Fig 7.
Mitochondrial respiration in T and B cells from healthy lean donors.
(A) OCR and (B) ECAR Mito Stress test Seahorse profiles for resting T and B cells from the same healthy individuals (N = 3). (C) Change in ECAR after addition of oligomycin. Data is the average of measurements 6–8 from panel B minus measurements 3–5. (D) Area under the curve analysis of ECAR profile. (E) Metabolic parameters and (F) OCR:ECAR at basal and maximal respiration using SHORE. Differences in panels C-F are determined by paired two-tailed student’s t test.