magnetic separation of cd14+ and cd3+ cells and fluorespirometry to study mitochondrial dynamics

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

A cell&#39;s ability to function and survive in a period of physiological stress is largely dependent on its ability to meet the energetic requirement to restore homeostasis1,2. Energy demand rises in response to a variety of stimuli. For instance, increased muscle contraction during exercise increases the utilization of ATP and glucose by skeletal muscle, and a rise in protein synthesis following infection increases the utilization of ATP by immune cells for cytokine production and proliferation3,4,5,6. A spike in energy demand triggers a series of bioenergetic processes to restore the ATP/ADP ratio. As ATP is consumed, ADP levels rise and stimulate F1F0&#160;ATP-synthase (complex V), which requires a protonmotive force to drive its mechanical rotation and catalytic conversion of ADP to ATP within the mitochondrion7. The protonmotive force is an electrochemical gradient created by the pumping of protons during the transfer of electrons from substrates to oxygen through the electron transport system (ETS) within the inner mitochondrial membrane. The resulting difference in proton concentration (delta pH) and electrical potential (membrane potential) creates the protonmotive force that drives ATP synthesis and oxygen consumption in response to energy demand reducing the ATP/ADP ratio or raising ADP levels. The affinity of mitochondria to ADP can be determined by the calculation of the Km or EC50 of ADP-stimulated respiration of isolated mitochondria or permeabilized cells8,9. This method has shown that permeabilized muscle fibers from older humans require a greater concentration of ADP to stimulate 50% of their maximal oxidative phosphorylation capacity than those of younger subjects9. Similarly, aging mouse skeletal muscle requires more ADP to lower the production of mitochondrial reactive oxygen species (ROS)10,11. Additionally, ADP sensitivity is reduced in permeabilized muscle fibers of mice with diet-induced obesity relative to controls and is enhanced in the presence of insulin and following nitrate consumption12,13. Thus, the capacity of mitochondria to respond to energy demand varies under different physiological conditions, but this has not been previously explored in the context of immune cells.
Peripheral blood mononuclear cells (PBMCs) are commonly used to investigate cellular bioenergetics in human subjects14,15,16,17,18,19,20. This is largely due to cells being easily obtainable from uncoagulated blood samples in clinical studies, the responsiveness of cells to metabolic perturbations, and the methods developed by various groups to interrogate mitochondrial metabolism by using inhibitors and uncouplers to determine the maximal and minimal capacity of mitochondrial respiration21,22. These methods have led to an appreciation of the roles of bioenergetics in aging, metabolic disease, and immune function14,20,23,24. Mitochondrial respiratory capacity is often reduced in skeletal muscle and PBMCs under conditions of heart failure18,25. PBMC bioenergetics are also correlated with cardiometabolic risk factors in healthy adults17 and are responsive to treatments such as nicotinamide riboside18. PBMCs include neutrophils, lymphocytes (B-cells and T-cells), monocytes, natural killer cells, and dendritic cells, which all contribute to PBMC mitochondrial capacity26,27,28. In addition, cellular bioenergetics play a crucial role in immune cell activation, proliferation, and renewal23. However, a limitation of these methods is that the cells are not functioning under a physiological range of substrates. Additional methods are therefore required to interrogate mitochondrial function in substrate concentrations that are more relevant to what cells experience in vivo.
Mitochondrial membrane potential (mMP) is the major component of a protonmotive force and is essential for a variety of mitochondrial processes beyond ATP production, such as regulation of respiratory flux, reactive oxygen species production, protein and ion import, autophagy, and apoptosis. mMP can be assessed with electrochemical probes or fluorescent dyes sensitive to changes in membrane polarization like JC-1, Rhod123, DiOC6, tetramethyl rhodamine (TMRE) or methyl ester (TMRM), and safranin. The latter two are lipophilic cationic dyes that have been successfully used in high-resolution fluorespirometry of tissue homogenates, isolated mitochondria, and permeabilized tissue11,29,30,31,32,33. In this technique, TMRM is used in quench mode, where cells are exposed to a high concentration of TMRM that accumulates in the mitochondrial matrix when polarized (high mMP and protonmotive force), resulting in the quenching of cytosolic TMRM fluorescence. When mitochondria depolarize in response to ADP or uncouplers, the dye is released from the matrix, increasing the TMRM fluorescent signal34,35. The purpose of this method is to simultaneously measure changes in mitochondrial respiration and mMP in response to ADP titrations in human-derived PBMCs, circulating monocytes, and T-cells, and it can also be applied to mouse splenic T-cells.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

For a cell to function and survive following a period of physiological stress, it needs to be able to meet the energy demand required to restore homeostasis. In our lab, we seek to identify how mitochondria respond and adapt to nutritional stressors to better understand how mitochondria mediate the risk of developing disease. The current methods for testing PBMC bioenergetics involve measuring respiratory capacity after adding inhibitors and uncouplers.These methods have helped us understand significant bioenergetic changes in PBMCs in different disease states. Membrane potential is essential for ATP synthesis and regulates processes such as respiratory flux, reactive oxygen species, and autophagy. However, we still have limited knowledge about how mitochondrial respiration and membrane potential together respond to physiological substrate concentrations in PBMCs.The advantage of this technique is that it allows for an integrated analysis of mitochondrial membrane potential and oxygen consumption in human PBMCs in response to increasing levels of ADP. This method allows for the quantification of the sensitivity of mitochondria to a shift in energy demand.

Magnetic Separation of CD14+ and CD3+ Cells and Fluorespirometry to Study Mitochondrial Dynamics

magnetic-separation-cd14-cd3-cells-fluorespirometry-to-study

Research

JoVE Journal

Biology

214 visualizações.  University of Washington. Para começar, instale câmaras de 0,5 mililitro no respirômetro O2K de acordo com as instruções do fabricante. Ligue o instrumento e conecte-o ao software fornecido para aquisição de dados. Lave as câmaras três vezes com água deionizada.Substitua a água por 0,54 mililitros de MiR05 e feche totalmente as rolhas. Depois de remover o excesso de tampão, levante as rolhas para permitir que o oxigênio ambiente se equilibre com o oxigênio da câmara. Coloque uma coluna no campo m...