Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids

Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis

Synthetic peptides incorporating well-folded β-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG β-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the β-hairpin-conjugated substrate and its PKCα-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity

Fast-lysis cell traps for chemical cytometry

Electrically addressable cell traps were integrated with capillary electrophoresis for the analysis of the contents of single adherent cells. Electrodes composed of indium tin oxide were patterned on a glass surface followed by formation of topographical cell traps using 1002F photoresist. Single cells trapped in the holes could be lysed in less than 66 ms by applying a brief electric field (10 ms) across the electrode beneath the cell and the ground electrode placed in the aqueous media above the cell traps. The gas formed during cell lysis remained localized within the cavity formed by the 1002F photoresist. The retention of the gas in the cell trap enabled the cell traps to be coupled to an overlying capillary without blockage of the capillary. Single cells cultured in the traps were loaded with fluorescein and Oregon Green and then electrically lysed. By simultaneous application of an electric field to the capillary, the cell’s contents were loaded into the capillary and electrophoretically separated. Orgeon Green and fluorescein from a single cell were fully resolved in less than two minutes. The use of a single patterned electrode beneath the 1002F cell trap yielded a simple easily fabricated design that was robust when immersed in aqueous solutions. Moreover, the design can easily be scaled up to create arrays of adherent cells for serial analyses using a single capillary or for parallel analysis by mating to an array of capillaries. Enhancing the rate of analysis of single adherent cells would enable a greater understanding of cellular physiology

Characterization of the laser-based release of micropallets from arrays

The micropallet array system uses a pulsed laser to release pallets tens of microns to hundreds of microns in size from a larger array, enabling selective isolation of single cells adherent to the pallets. In this study, the laser-based release of pallets was characterized with respect to pallet array and laser parameters. The threshold laser energy required for pallet release increased linearly with the area of the pallet in contact with the underlying glass substrate. The spacing of the pallets within an array as well as the thickness or height of the pallet did not impact the energy required to release a pallet. Delivery of multiple laser pulses decreased the energy/pulse required for pallet release when the pallets were 100 microns or greater on a side. In addition to the square pallets, complex structures such as cantilevers and spirals could be released without damage using the pulsed laser. Identification of the pallet-array variables influencing the energy required for pallet release as well as strategies to minimize this energy will prove critical in optimizing the release of pallets with cells on the arrays

Photoresist with Low Fluorescence for Bioanalytical Applications

The negative photoresist SU-8 has found widespread use as a material in the fabrication of microelectrical-mechanical systems (MEMS). While SU-8 has been utilized as a structural material for biological MEMS, a number of SU-8 properties limit its application in these bioanalytical devices. These attributes include its brittleness, nonspecific adsorption of biomolecules, and high fluorescence in the visible wavelengths. In addition, native SU-8 is a poor substrate for cellular adhesion. Photoresists composed of resins with epoxide side groups and photoacids were screened for their ability to serve as a low fluorescence photoresist with sufficient resolution to generate microstructures with dimensions of 5-10 μm. The fluorescence of structures formed from 1002F photoresist (1002F resin combined with triarylsulfonium hexafluoroantimonate salts) was as much as 10 times less fluorescent than similar SU-8 microstructures. The absorbance of 1002F in the visible wavelengths was also substantially lower than that of SU-8. Microstructures or pallets with an aspect ratio as high as 4:1 could be formed permitting 1002F to be used as a structural material in the fabrication of arrays of pallets for sorting adherent cells. Several different cell types were able to adhere to native 1002F surfaces and the viability of these cells was excellent. As with SU-8, 1002F has a weak adhesion to glass, a favorable attribute when the pallet arrays are used to sort adherent cells. A threshold, laser-pulse energy of 3.5 μJ was required to release individual 50-μm, 1002F pallets from an array. Relative to SU-8, 1002F photoresist offers substantial improvements as a substrate in bioanalytical devices and is likely to find widespread use in BioMEMS

Large-Area Silicon Detectors for the Advanced Composition Explorer (ACE) Solar Isotope Spectrometer (SIS)

Extensive measurements were made of the thicknesses and dead-layers of the large-area, highpurity silicon detectors used for the Solar Isotope Spectrometer (SIS), an instrument to be launched on the Advanced Composition Explorer (ACE) spacecraft. Tests using accelerated beams of heavy nuclei were also carried out to characterize the completed instrument

The ACE-CRIS Scintillating Optical Fiber Trajectory (SOFT) Detector: Calibrations at the NSCL and GSI

The Scintillating Optical Fiber Trajectory (SOFT) detector, the hodoscope for the Cosmic Ray Isotope Spectrometer (CRIS) on the NASA Advanced Composition Explorer, was calibrated using 155 MeV/n He, Li, C, N, 0, and Ar at the Michigan State University National Superconducting Cyclotron Laboratory (NSCL), and 200 - 700 MeV/n C, Si, and Fe at the GSI facility in Darrnstadt. Germany. The flight instrument consists of three hodoscope fiber planes and one trigger plane. read out by an image intensified CCD camera system and by intensified photodiodes respectively. The spatial and angular resolution of the hodoscope is described, along with the detection efficiency of both the hodoscope and trigger plane as a function of charge

Calcium Signals Driven by Single Channel Noise

Usually, the occurrence of random cell behavior is appointed to small copy numbers of molecules involved in the stochastic process. Recently, we demonstrated for a variety of cell types that intracellular Ca2+ oscillations are sequences of random spikes despite the involvement of many molecules in spike generation. This randomness arises from the stochastic state transitions of individual Ca2+ release channels and does not average out due to the existence of steep concentration gradients. The system is hierarchical due to the structural levels channel - channel cluster - cell and a corresponding strength of coupling. Concentration gradients introduce microdomains which couple channels of a cluster strongly. But they couple clusters only weakly; too weak to establish deterministic behavior on cell level. Here, we present a multi-scale modelling concept for stochastic hierarchical systems. It simulates active molecules individually as Markov chains and their coupling by deterministic diffusion. Thus, we are able to follow the consequences of random single molecule state changes up to the signal on cell level. To demonstrate the potential of the method, we simulate a variety of experiments. Comparisons of simulated and experimental data of spontaneous oscillations in astrocytes emphasize the role of spatial concentration gradients in Ca2+ signalling. Analysis of extensive simulations indicates that frequency encoding described by the relation between average and standard deviation of interspike intervals is surprisingly robust. This robustness is a property of the random spiking mechanism and not a result of control