Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

How to build an inexpensive cyclotherm instrument for automated polymerase chain reaction

The Cyclotherm instrument is a functionally fully equivalent but inexpensive alternative to commercial instruments for automated polymerase chain reaction (PCR). It can be rebuilt under conditions of a biochemical laboratory for less than +1000. A Peltier element is used for heating and cooling of the reaction vials and the temperature and timing of the PCR cycles are controlled by a BASIC program in a SHARP PC 1600 low cost computer.     

Fluorometric microbore amino acid analyzer: the construction of an inexpensive, highly sensitive instrument using o-phthalaldehyde as a detection agent.

The construction of a highly sensitive microbore amino acid analyzer is described. It is based on a standard column chromatographic separation technique with fluorometric detection utilizing o-phthalaldehyde. This analyzer has several desirable features: low column chromatographic pressure, high sensitivity, and easy maintenance. Good precision at a level of 10 pmol is obtained and as little as 0.2 μg protein has been hydrolyzed for composition analysis. It incorporates the use of the single-column method and constant molarity buffers to shorten the analysis time. It is fully automatic and capable of analyzing 130 samples within 7 days with attention required only for reloading 20 samples while the instrument is in operation. Amino acid composition determination based on the peak area and the peak height is discussed.     

An inexpensive instrument for sediment erosion-accumulation rate measurement in intertidal environments

A low-cost instrument to determine elevation changes in intertidal environments is presented. The instrument consists of two small PVC pipes driven into the soil and a ruler attached to a bar sustained by two rods that are inserted into the pipes during the measurement. This method cost only a fraction of similar ones described in the literature. Furthermore, because large numbers can be deployed in an area, statistically significant sedimentation-erosion rates can be obtained for places where spatial variances are very large.     

Gear modifications reduced humpback whale entanglements in a commercial rock lobster fishery

Entanglement of whales in fishing gear occurs globally and where populations are recovering from past exploitation, entanglement frequency is likely to increase. The Western Australian population of humpback whales (Megaptera novaeangliae) is growing rapidly, yet from 1990 to 2010 the number of whales reported entangled in gear from the pot-based western rock lobster fishery was relatively stable at around one per year. However, from 2010, reported entanglements increased, reaching a maximum of 17 in 2013. This increase occurred immediately after a shift to a year-round quota-based fishery that eliminated the annual 4½-month closure that coincided with the whale migration. Gear modifications that eliminated surface rope, shortened rope lengths, and reduced float numbers were implemented in June 2014 to reduce whale entanglements. The effectiveness of these modifications was evaluated using a Bayesian model that incorporated changes in humpback whale population size, entanglement reporting probability, fishing effort, and whale migration timing. Our analyses indicate that gear modifications reduced entanglement in fishing gear from the rock lobster fishery by at least 25% (with 95% probability), with a median reduction of 64%. The model also showed that the greatest entanglement risk occurs on the northward migration and in water depths of 55–73 m.     

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

Characterization of the sensitivity of side scatter in a flow-stream waveguide flow cytometer.

BACKGROUND: We previously reported a new optical configuration, in which both the side scatter and the fluorescence are collected using the index-guided, total internal reflection of a flow stream in air (the flow-stream waveguide). METHODS: Using a mixture of 0.202-microm and 0.093-microm diameter polystyrene beads, we have characterized the side scatter (SSC) sensitivity of a custom-built flow cytometer (miniFlo) which incorporates a flow-stream waveguide. RESULTS: The SSC-triggered SSC signal of 0.093-microm polystyrene beads in water was almost baseline resolved from the background. We also measured the SSC-triggered SSC signal of the same beads in water on our FACScan, which is a commercial unit with the conventional optical arrangement that uses a custom imaging objective to collect light from a sheath flow cuvette in perpendicular direction-the signal from 0.093-microm beads was not resolved from the background. CONCLUSIONS: The SSC sensitivity of miniFlo is one of the best reported in the literature. Cytometry 37:160-163, 1999. Published 1999 Wiley-Liss, Inc.     

Precise temperature control of the Gilford spectrophotometer: a simple and inexpensive modification.

A method has been developed for the preparation of defined-length homopolymers of long chain length in high yield.     

Flow cytometry controls, instrument setup, and the determination of positivity.

A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.     

Routine amino acid capillary column chromatography of microquantities in the picomole range--procedures and modifications of a commercial analyzer

A commercial micro amino acid analyzer using capillary columns (internal diameter 0.7 mm) had to be modified greatly in order to get satisfactory separation and quantitative analysis on the microscale of free amino acids in biological fluids, tissue extracts, and hydrolysates. Furthermore, the usual analytical conditions were changed (i.e., lowering of the buffer pH) and a procedure and apparatus for sample deproteinization is described which allows simple handling of volumes in the range from 20 to 100 μl. The modifications described guarantee better and more reproducible analyses than reported so far. A design of a new “analytical unit” for capillary column chromatography is proposed.     

Histone modifications and nuclear architecture: a review.

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.     

Nine fluorescence parameter analysis on a four-color fluorescence activated flow cytometer.

BACKGROUND: In many cases a frequent monitoring of blood is important, e.g. during an infection. Often the availability of blood is the limiting factor. METHODS: 50 microl blood were stained and analyzed using a standard four-color cytometer. Percentages of leukocytes were calculated by FlowJo software. RESULTS: Our protocol allows the differentiation of B cells, T cells, NK cells, neutrophils, and monocytes/macrophages in small volumes of blood. CONCLUSIONS: Using nine fluorochrome-labeled antibodies and a specific gating strategy we were able to differentiate the main immune cells in minute amounts using one staining step.     

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.