In vitro stimulation of bovine peripheral blood lymphocytes: comparison of round- and flat-bottom microtiter plates for detection of tuberculin hypersensitivity

Sloane, E. D.; Muscoplat, Charles C; Kaneene, J. M.; Klausner, D. J.; Thoen, Charles O.; Johnson, Donald W.

doi:10.1128/jcm.7.2.172-175.1978

J Clin Microbiol

1978

DOI: 10.1128/jcm.7.2.172-175.1978

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In vitro stimulation of bovine peripheral blood lymphocytes: comparison of round- and flat-bottom microtiter plates for detection of tuberculin hypersensitivity

E. D. Sloane¹,

Charles C Muscoplat²,

J. M. Kaneene³

et al.

Abstract: Lymphocytes from Mycobacterium bovis-sensitized and normal cattle were cultured in round- and/or flat-bottom microtiter plates and stimulated with M. bovis purified protein derivative (PPD) tuberculin. Blastogenic responses of lymphocytes from M. bovis-sensitized cattle to PPD cultured in round-bottom plates were significantly greater than those of lymphocytes cultured in flat-bottom microtiter plates. Normal lymphocytes of nonsensitized cattle were not stimulated by PPD in either round- or flat-bottom microti… Show more

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Cited by 2 publications

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Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial

Gates

Griffiths

2018

Immunol Res

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Despite rabbits becoming an increasingly popular animal model, a flow cytometry panel that combines T cell markers (CD3, CD4, CD8, CD25, FOXP3) with a method for monitoring proliferation is lacking in this species. It has been shown that the rabbit model can be used to identify xenoantigens within bovine pericardium (BP), a common biological heart valve replacement material; however, these methods rely on monitoring the humoral immune response. The development of a rabbit T cell proliferation assay has utility in monitoring graft-specific cell-mediated immune responses toward bovine pericardium. Isolation and culture conditions were optimized to avoid cell death, red blood cell contamination, and non-specific proliferation. Effect of cell culture and stimulation on distribution and intensity of T cell markers was analyzed and compared between cells isolated from naïve and BP-immunized rabbits. Submaximal levels (0.25 μg/mL) of concavalin A were used to stimulate proliferation toward BP extract, with resultant proliferation compared between naïve and BP-immunized rabbits. Density stratification followed by ammonium potassium chloride (ACK) lysis yielded the greatest number of viable peripheral blood mononuclear cells with the least amount of erythrocyte contamination. Flat-bottomed plates were necessary to reduce non-specific proliferation in culture. T cells responded appropriately to maximal mitogenic stimulation (5 μg/mL concavalin A). Interestingly, immunization increased the intensity of FOXP3 in T regulatory cells compared to cells from naïve animals. With addition of submaximal levels of concavalin A, T cells from immunized rabbits proliferated in response to BP protein extract, while cells from naïve rabbits did not. In immunized rabbits, not only did more CD4 T cells proliferate in response to BP re-stimulation, but the intensity of CD25 was increased indicating cell activation. This research provides a functional cell-mediated screening assay for assessment of BP-based biomaterials in rabbits, overcoming the limitations of previous humoral immune system-based assessments of biomaterial antigenicity in this important experimental animal species.

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Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial

Gates

Griffiths

2018

Immunol Res

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Cell mediated and humoral immune response of chickens to live infectious bronchitis vaccines

Timms

Bracewell

1981

Research in Veterinary Science

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In vitro stimulation of bovine peripheral blood lymphocytes: comparison of round- and flat-bottom microtiter plates for detection of tuberculin hypersensitivity

Cited by 2 publications

References 14 publications

Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial

Chronic graft-specific cell-mediated immune response toward candidate xenogeneic biomaterial

Cell mediated and humoral immune response of chickens to live infectious bronchitis vaccines

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