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Biomedical subjects

K R Harkins

Publications and source records attributed to K R Harkins.

6 recordsLinked to original sources
PubMed2000

Antigen-specific proliferation of porcine CD8alphaalpha cells to an extracellular bacterial pathogen.

A vaccine inducing protective immunity to a spirochaete-induced colitis of pigs predominantly stimulates expansion of CD8+ cells in vivo and in antigen-stimulated lymphocyte cultures. CD8+ cells, however, are rarely considered necessary for protection against extracellular bacterial pathogens. In the present study, pigs recovering from colitis resulting from experimental infection with Brachyspira (Serpulina) hyodysenteriae had increased percentages of peripheral blood CD4- CD8+ (alphaalpha-expressing) cells compared with non-infected pigs. CD8alphaalpha+ cells proliferated in antigen-stimulated cultures of peripheral blood mononuclear cells from B. hyodysenteriae-vaccinated pigs. Proliferating CD8alphaalpha+ cells consisted of CD4-, CD4+ and gammadelta T-cell receptor-positive cells. CD4- CD8alphabeta+ cells from vaccinated or infected pigs did not proliferate upon in vitro antigen stimulation. Of the CD8alphaalpha cells that had proliferated, flow cytometric analysis indicated that the majority of the CD4+ CD8+ cells were large (i.e. lymphoblasts) whereas the CD4- CD8+ cells were predominantly small. Addition of monoclonal antibodies (mAb) specific for either porcine major histocompatibility complex (MHC) class I or class II antigens diminished B. hyodysenteriae-specific proliferative responses whereas addition of mAb to porcine MHC II, but not porcine MHC I, reduced the CD8alphaalpha response. In vitro depletion of CD4+ cells by flow cytometric cell sorting diminished, but did not completely abrogate, the proliferative response of cells from vaccinated pigs to B. hyodysenteriae antigen stimulation. These results suggest that CD8alphaalpha cells are involved in recovery and possibly protection from a spirochaete-induced colitis of pigs; yet, this response appears to be partially dependent upon CD4+ cells.

Animals↗
PubMed1994

In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain.

A rapid and sensitive assay is described for the determination of cell viability of adherent and nonadherent cells that can be performed in situ in 96-well microtiter plates using fluorescence plate scanners. The assay, based on dye exclusion, utilizes a plasma membrane-impermeable, dimeric cyanine dye (YOYO-1). YOYO-1 fluoresces brightly only when bound to nucleic acids. Cells are incubated with YOYO-1, and fluorescence is measured before and after the addition of detergent, which allows the dye to enter the cells. The fluorescence before detergent treatment originates from nonviable cells that have membrane damage and take up YOYO-1. The fluorescence after detergent treatment originates from all cells in the sample. The ratio of the two fluorescence values is used as an indicator of cell viability. The cell viability results of this microplate assay closely resemble those of dye exclusion studies by flow cytometry and are similar but not identical to those of the thiazolyl blue assay, which uses a metabolic indicator of cell death. Because the assay can be performed in situ, without removing the medium, disintegrated cells, cell aggregates, and cells that stick to culture vessel walls are all included in the measurement.

Benzoxazoles↗
PubMed1990

Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts.

We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.

Base Sequence↗
PubMed1988

Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts.

We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.

Cell Separation↗
PubMed1987

Factors governing the flow cytometric analysis and sorting of large biological particles.

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.

Cell Separation↗