However, larger double-blind clinical trials to confirm this belief are awaited

described. Briefly, MedChemExpress R115777 Glucose uptake was measured by incorporation of 2-deoxy-dglucose. Glycolytic flux was determined by measuring the detritiation of glucose. Glucose, glutamine, and pyruvate oxidation was measured by culture of cells in U-14C glucose, glutamine, and pyruvate respectively to measure production of 14CO2. 3Hpalmitic acid was used to measure lipid oxidation by the production of 3H2O. Oxygen consumption rate and extracellular acidification rate were measured with a XF24 extracellular flux analyzer. Briefly, 1.4106 unstimulated or 106 stimulated cells per well were seeded in a Cell-Tak coated plate, and OCR and ECAR measurements were normalized to cell number. Cells were initially plated in XF Seahorse media with glutamine alone when glucose was injected in ECAR tests, or J Immunol. Author manuscript; available in PMC 2015 April 15. Caro-Maldonado et al. Page 5 both glucose and glutamine in mitochondrial stress test using the following concentrations of injected compounds as indicated in the text: Oligomycin 1M, Rotenone, electron transport chain accelerator FCCP, Antimycin A, 2-DG, glucose. Immunizations and ELISAs Some mice were immunized by intraperitoneal injection with 20g of Ovalbumin coupled to 4-hydroxy-3-nitrophenyl in alum. At various time points serum was collected and examined by ELISA for total and anti- acetyl-bovine serum albumin IgM and IgG in a heterocritic antibody response for NP-reactive antibodies, as described. NIP5 and NIP25 indicate low and high levels of NIP coupling to BSA and reflect high affinity anti-NP and total anti-NP responses, respectively. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Results B cell metabolic reprogramming upon activation T cells transition from predominantly oxidative metabolism to aerobic glycolysis upon activation. B cells have also been shown to also increase glycolysis upon stimulation, although the regulation of this process is less characterized. To examine the metabolic regulation of B cells, metabolic parameters of isolated B cells were measured ex vivo or after six hours stimulation with LPS. At this time point, B cells have not entered the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844160 cell cycle and metabolic changes are not due to potential secondary effects of proliferation. Extracellular flux analysis showed that six hours of LPS was sufficient to significantly increase B cell extracellular acidification rate in response to glucose compared to that of B cells examined ex vivo. ECAR likely represents glycolytic flux to lactate production and was further increased to the maximal glycolytic capacity upon addition of the F1/F0 ATPase inhibitor, oligomycin, to suppress mitochondrial ATP production. This increase in lactate was dependent on glucose flux through glycolysis, as ECAR was sharply reduced by the glycolytic inhibitor 2-DG. Glycolytic flux was also measured directly using radiolabeled glucose in an assay that incorporates glucose uptake and glycolytic flux through enolase. In agreement with ECAR, 24 hours of LPS treatment significantly increased glycolytic rate. Glucose can also be oxidized through the pentose phosphate pathway or the mitochondrial tricarboxylic acid cycle and LPS stimulation led to a significant increase in glucose oxidation. Oxidative phosphorylation can yield large amounts of ATP and is fueled by glucose, glutamine, or lipid metabolism. Like glycolysis, the oxygen consumption rate of B cells increased following B cell stimulation. Both basal OCR and ma