Microfluidic devices for culturing primary mammalian neurons at low densities

Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for > or = 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to < or = 2 days. When the closed-channel PDMS devices are extracted, biocompatibility improves allowing for reliable neuron cultures at low densities for > or = 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for > or = 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.     

The effect of pressure on the crystalline electric field levels of superconducting La1−xTbxAl2

The pressure dependence of the superconducting transition temperature (dT_c/dP) is smaller for La_1?xTb_xAl₂ than for LaAl₂. The difference is attributed to an increase with pressure of the crystalline electric field energies experienced by the Tb ion.     

A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis

Single-cell measurements allow a unique glimpse into cell-to-cell heterogeneity; even small changes in selected cells can have a profound impact on an organism's physiology. Here an integrated approach to single-cell chemical sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydroxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10(-14) mol to 10(-16) mol, an order of magnitude higher than observed in untreated pinealocytes.     

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.     

Tor gives the inverse to the Hilbert function of a graded algebra

The Hilbert generating function of Tor^U(A, A) gives the inverse of the Hilbert generating function of U where U is a graded A algebra.     

Separation of amino acid and peptide stereoisomers by nonionic micelle-mediated capillary electrophoresis after chiral derivatization

Enantiomers of amino acids and peptides were derivatized with a fluorescent chiral reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [R-(−)- or S-(+)-NBD-PyNCS] and the resulting diastereomeric derivatives separated by capillary electrophoresis (CE). The CE running buffer consisted of 25 mM acetate buffer (pH 4) and 10 mM of the nonionic surfactant Triton X-100. The excitation maximum of NBD-PyNCS at 480 nm matches the major Ar-ion emission line at 488 nm allowing sensitive laser-induced fluorescence detection with limits of detection around 50 nM.

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-Proline and

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-aspartate spiked (at 10⁻⁴ M and 10⁻⁵ M concentrations, respectively) into complex biological matrices (rabbit serum and homogenate of Aplysia californica buccal ganglion) are detected without matrix interferences. This method has also been applied to the determination of

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- and

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-amino acid residues in peptides after acid hydrolysis. Results from the chiral analysis of the naturally-occurring peptide, gramicidin D, are shown.     

Two classical splitting theorems: Easy proofs

The two main splitting theorems for a division ring which is finite dimensional over its center are obtained as corollaries to:Lemma. Suppose B ? A ? D where B is a (commutative) subfield of the division ring D and A is an intermediate ring. (For any set X ? D let X′ denote the cenralizer of X in D.) If B = B″, then

$\text{A}{}_{\mathrm{?B}}\text{D}\text{→}\text{π}\text{Hom}{}_{\mathrm{A\prime }}\text{(B,D)}$

where

$\text{π(a ? d) = ab′d, a ∈ A, d ∈ D}\text{and}\text{B′ ∈ B′,}$

is injective.     

Effects of Adsorbed Polyaniline on Redox Processes on As(2)O(3) Surfaces

Polyaniline deposited on As(2)O(3) surface resulted in a new material, which was characterized by infrared spectoscopy, thermogravimetry, differential scanning calorimetry, scanning electron microscopy, X-ray diffraction, and cyclic voltammetry. The mass percentage of polymer deposited on oxide surface is approximately 13%. The scanning electron microscopy images as well as the X-ray diffraction patterns provided conclusive evidence that the oxide surface is coated by the polymer. The cyclic voltammograms of the polyaniline adsorbed on As(2)O(3) surface showed that the adsorbate exerts remarkable effects on redox processes on this oxide. The pure oxide exhibited two oxidation/reduction peaks at 0.25/-0.06 and 0.47/-0.25 V attributed tentatively to the processes As(2)O(3)(s)+6H(+)+6e(-)=2As(s)+3H(2)O and As(s)+3H(+)+3e(-)=AsH(3)(g), respectively. The polyaniline-coated sample exhibited a better-defined voltammogram in which the first oxidation peak of the oxide had its intensity increased about four times. Copyright 2000 Academic Press.     

A multilayer poly(dimethylsiloxane) electrospray ionization emitter for sample injection and online mass spectrometric detection

An ESI emitter made of poly(dimethylsiloxane) interfaces on-chip sample preparation with MS detection. The unique multilayer design allows both the analyte and the spray solutions to reside on the device simultaneously in discrete microfluidic environments that are spatially separated by a polycarbonate track-etched, nanocapillary array membrane (NCAM). In direct spray mode, voltage is applied to the microchannel containing a spray solution delivered via a syringe pump. For injection, the spray potential is lowered and a voltage is applied that forward biases the membrane and permits the analyte to enter the spray channel. Once the injection is complete, the bias potential is switched off, and the spray voltage is increased to generate the ESI of the injected analyte plug. Consecutive injections of a 10 microM bovine insulin solution are reproducible and produce sample plugs with limited band broadening and high quality mass spectra. Peptide signals are observed following transport through the NCAM, even when the peptide is dissolved in solutions containing up to 20% seawater. The multilayer emitter shows great potential for performing multidimensional chemical manipulations on-chip, followed by direct ESI with negligible dead volume for online MS analysis.