Chris Hartnett

R&D Systems, Minneapolis, Minnesota, United States

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Publications (4)5.75 Total impact

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    ABSTRACT: Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas both stimulatory and inhibitory effects were observed on IFNγ secretion. Our data demonstrate a large dynamic range in IFNγ and IL-4 secretion among different donors, which may reflect animal health and serve as a valuable diagnostic marker.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 792:39-45. · 1.29 Impact Factor
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    ABSTRACT: Peripheral blood mononuclear cells (PBMCs) were obtained from 6 foals <1 week of age, 6 foals between 3 and 4 months of age, and 10 adult horses. PBMCs were stimulated with concanavalin A (ConA) or calcium ionomycin-phorbol myristate acetate (CaI-PMA) and the frequency of interferon IFN-gamma and IL-4 secreting cells was measured using an equine-specific ELISPOT assay. The number of IFN-gamma secreting cells was significantly lower in both groups of foals than in adult horses regardless of the mitogen used for stimulation. The number of IFN-gamma secreting cells was significantly higher in cells stimulated with CaI-PMA than in cells stimulated with ConA. In cells stimulated with CaI-PMA, the number of IL-4 secreting cells was significantly lower in both groups of foals compared to adult horses. In adult horses only, CaI-PMA stimulation resulted in significantly more IL-4 secreting cells than ConA stimulation. Regardless of age, the ratio of IFN-gamma/IL-4 spot forming cells (SFC) was significantly higher in cells stimulated with CaI-PMA than in cells stimulated with ConA. These findings indicate that the frequency of IFN-gamma and IL-4 secreting cells is lower in foals than in adult horses and that the type of mitogen used has a profound effect on the relative production of both cytokines.
    Veterinary Immunology and Immunopathology 07/2009; 133(1):66-71. · 1.88 Impact Factor
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    ABSTRACT: Enzyme-linked immunospot (ELISPOT) assays are widely used as a technique that allows determining the frequency of cytokine-releasing cells. Colored spots appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. Porous membranes are used in ELISPOT plates to provide support for growing cells, thus making it difficult to remove them by washing. Cells that have adhered to the membrane may be stained nonspecifically, producing a background and then counted as specific spots. We have tested a cell detachment reagent, Accumax, and found that it may be used to remove a large number of cells adhered to the microplate membranes. Accumax was tested in 16 different ELISPOT assays, including human interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-13, IL-1beta, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha; mouse IL-4, IL-6, IFN-gamma, and TNF-alpha; rat IL-2 and IFN-gamma; and canine IFN-gamma. Accumax was found to be compatible with human IL-13, IL-1beta, IL-2, IL-4, IL-5, and IL-8 and mouse IL-4, IL-6, and TNF-alpha ELISPOT assays, allowing one to remove a large number of adhered cells without hindering ELISPOT assay performance. However, Accumax was incompatible with human IFN-gamma, mouse IFN-gamma, canine IFN-gamma, and rat IFN-gamma ELISPOT assays because Accumax reduced the intensity of staining and the number of spots formed.
    Methods in molecular biology (Clifton, N.J.) 02/2005; 302:87-94. · 1.29 Impact Factor
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    ABSTRACT: Living in the era of multiplex detection systems, it appears attractive to develop enzyme-linked immunospot (ELISPOT) assays for the detection of more than one cytokine released by the same cell. However, despite technical simplicity in building such an assay, several factors have to be considered when designing multiplex ELISPOT assays. We have used four capture antibodies (hIFN-gamma, hIL-2, hIL-4, and hTNF-alpha) either in combination or individually to coat polyvinylidene difluoride membrane-backed Millipore 96-well plates. Several cell stimulations were also used, including Concanavalin A, Phorbol Myristate Acetate (PMA) and calcium ionophore (CaI), phytohemagglutinin, CD3e, and lipopolysaccharide. Biotinylated antibodies were used either individually or combined together to detect secreted cytokines. We have found that when plates were coated with all four capture antibodies and captured cytokines were detected using either one detection antibody or all four detection antibodies combined together, fewer spots could be seen when compared with a plate coated with a single capture antibody followed by using its matched detection antibody counterpart. Interestingly, negative interferences between antibodies were less profound when detection antibodies rather than capture antibodies were mixed together.
    Methods in molecular biology (Clifton, N.J.) 02/2005; 302:273-88. · 1.29 Impact Factor