Phagosomal chloride dynamics in the alveolar macrophage.

Acidification in intracellular organelles is tightly linked to the influx of Cl- counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl- transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl- concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl- influx utilized predominantly the Cl- channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl- content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl- during phagosomal particle uptake. Cl- -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease.

Real time imaging of single extracellular vesicle pH regulation in a microfluidic cross-flow filtration platform.

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.

Microfluidic platform generates oxygen landscapes for localized hypoxic activation.

An open-well microfluidic platform generates an oxygen landscape using gas-perfused networks which diffuse across a membrane. The device enables real-time analysis of cellular and tissue responses to oxygen tension to define how cells adapt to heterogeneous oxygen conditions found in the physiological setting. We demonstrate that localized hypoxic activation of cells elicited specific metabolic and gene responses in human microvascular endothelial cells and bone marrow-derived mesenchymal stem cells. A robust demonstration of the compatibility of the device with standard laboratory techniques demonstrates the wide utility of the method. This platform is ideally suited to study real-time cell responses and cell-cell interactions within physiologically relevant oxygen landscapes.

Enhanced loading of Fura-2/AM calcium indicator dye in adult rodent brain slices via a microfluidic oxygenator.

A microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, our microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. This technique will facilitate opportunities for the study of calcium signaling of aging and long term stress related diseases. Moreover, it should be applicable to other membrane-permeant physiological indicator varieties.

Precise spatial and temporal control of oxygen within in vitro brain slices via microfluidic gas channels.

The acute brain slice preparation is an excellent model for studying the details of how neurons and neuronal tissue respond to a variety of different physiological conditions. But open slice chambers ideal for electrophysiological and imaging access have not allowed the precise spatiotemporal control of oxygen in a way that might realistically model stroke conditions. To address this problem, we have developed a microfluidic add-on to a commercially available perfusion chamber that diffuses oxygen throughout a thin membrane and directly to the brain slice. A microchannel enables rapid and efficient control of oxygen and can be modified to allow different regions of the slice to experience different oxygen conditions. Using this novel device, we show that we can obtain a stable and homogeneous oxygen environment throughout the brain slice and rapidly alter the oxygen tension in a hippocampal slice. We also show that we can impose different oxygen tensions on different regions of the slice preparation and measure two independent responses, which is not easily obtainable with current techniques.