Figure 3.

Increase in glycolysis persists at 24 hours but oxidative phosphorylation is no longer decreased in adult MDM. PBMC were isolated from buffy coats or from umbilical cord blood samples taken immediately following delivery. Adult or cord blood MDM were adherence purified for 7 days in 10% human serum. MDM were washed and detached from the plates by cooling and gentle scraping, counted and re-seeded on Seahorse culture plates prior to analysis in the Seahorse XFe24 Analyzer. Mtb (iH37Rv; MOI 1-10) or LPS (100 ng/ml) were added 24 hours prior to mitochondrial stress test analysis for adult (A) and cord blood (E) MDM. Differences in cell density was corrected for by crystal violet normalization. The extracellular Acidification Rates (ECAR) and Oxygen Consumption Rate (OCR) were recorded approximately every 9 minutes. The baseline ECAR, OCR and the ECAR/OCR ratio were analyzed for Mtb and LPS stimulated cells for the adult (B–D; n=7 line at median) and cord blood MDM (F–H; n=4 line at median). Comparison was made between the adult and cord OCR and ECAR/OCR ratio for the Mtb (I) and LPS (J) stimulated MDM. Graphs are expressed as % change from the third baseline reading of unstimulated cells. Statistically significant differences were determined using a paired (B, C, F, G) or unpaired (I, J) Student’s t test and one-way ANOVA (D, H), as appropriate; *P < 0.05, **P < 0.01, ***P < 0.001.