Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.
methyl ester; Dw m , mitochondrial membrane potential. AbstractPre-synaptic nerve terminals (synaptosomes) require ATP for neurotransmitter exocytosis and recovery and for ionic homeostasis, and are consequently abundantly furnished with mitochondria. Pre-synaptic mitochondrial dysfunction is implicated in a variety of neurodegenerative disorders, although there is no precise definition of the term 'dysfunction'. In this study, we test the hypothesis that partial restriction of electron transport through Complexes I and II in synaptosomes to mimic possible defects associated with Parkinson's and Huntington's diseases respectively, sensitizes individual terminals to mitochondrial depolarization under conditions of enhanced proton current utilization, even though these stresses are within the respiratory capacity of the synaptosomes when averaged over the entire population. We combine two novel techniques, firstly using a modification of a plate-based respiration and glycolysis assay that requires only microgram quantities of synaptosomal protein, and secondly developing an improved method for fluorescent imaging and statistical analysis of single synaptosomes. Conditions are defined for optimal substrate supply to the in situ mitochondria within mouse cerebrocortical synaptosomes, and the energetic demands of ion cycling and action-potential firing at the plasma membrane are additionally determined.
tained over macroscopic film thicknesses and large surface areas by vapor deposition of a single molecular species on an amorphous substrate. Inducing the non-centrosymmetric order by using selective hydrogen bonding to the substrate and between the molecules leads to a much better molecular ordering than with the technique described by Cai et al., [13] which requires control of the molecular beam direction relative to the substrate. The present technique does not require a well-defined molecular beam, and is also much simpler and faster than the synthetic methods used by Ulman [7] and Marks and Ratner [18] or for LB films.It is important to note that the alignment in our films is critically dependent on the ability of these molecules to selectively hydrogen bond to the surface, in order to begin the alignment process, and selectively hydrogen bond to itself in a unidirectional fashion in order to maintain the alignment over a large thickness. We have carried out preliminary experiments with molecules that only posses hydrogen bond acceptors and, therefore, can form hydrogen bonds to the substrate surface, but which lack hydrogen bond donors thus eliminating the possibility of intra-molecular hydrogen bonding, and have shown that only very thin films (of the order of a few monolayers thickness) with polar ordering perpendicular to the substrate surface can be grown. As the films grow thicker, we see that the ordering cannot be maintained.In conclusion, we have shown a simple and efficient new method to produce supramolecular self-assembled thin films with well-defined polar ordering perpendicular to the substrate surface. The resulting films are homogenous over a large area, and a regular, deterministic molecular ordering can be maintained over a large thickness of the order of 1 lm. The fact that the films were grown on amorphous glass substrates circumvents the need for surface epitaxy. The quality of the films and the simplicity of the fabrication method make this novel technique very attractive for the fabrication of nonlinear optical waveguides for frequency conversion or electrooptic modulation. Despite the relatively low nonlinearity (d 111 = 5 pm V ±1 ) that has been obtained in this first demonstration, the high degree of molecular ordering and the fact that the films consist of only one component make them potentially very interesting when compared to present candidates for organic electro-optic modulators, poled polymers. The higher degree of order and tighter molecular packing in our films should also lead to a better photostability when compared to amorphous systems with embedded chromophores. A further detailed investigation of the mechanism that leads to the molecular ordering and an optimization of the growth conditions should lead to a new generation of organic waveguides for frequency conversion and electro-optic modulation. Experimental.BITINPH was purchased from Opto-Organics Industrial Research Limited and purified by recrystallization from ethanol. The amorphous glass substrates were cl...
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