title: Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies.
abstract: The present study reports a method for isolating bovine colostrum mononuclear cells (CMC) for phenotyping and functional studies. As well as being an important source of immunoglobulins, colostrum also contains leukocytes that may be of greater importance for passive immunity than has previously been thought. Different protocols have been reported for isolating leukocytes from bovine colostrum, although none of these have been validated, and phenotypic analysis of cell populations has not always been performed. In this study, bovine CMC were isolated by density gradient centrifugation. Cell populations were identified by flow cytometry using antibodies against selected bovine cell surface markers and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. The mean cell count of isolated CMC was 3 × 10(4) and 1 × 10(5) per mL colostrum for the samples used in the flow cytometric assay and the proliferation assay, respectively. A mean of 25.4 ± 17.1% CMC were identified as T lymphocytes, 2.9 ± 3.0% as B lymphocytes and 32.7 ± 13.7% as macrophages. In terms of proliferation, the mean counts per minute were 4.3 × 10(3) and 1.8 × 10(4) for cells cultured in medium only or in the presence of concanavalin A, respectively, showing that CMC are viable and capable of responding to mitogen stimulation. Isolation of CMC and the subsequent phenotypic analysis of the different subpopulations were repeatable, with agreement indices varying between 0.5 and 1.0. Agreement indices for the proliferation assay were estimated at 0.8.
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Electronic address: vanessa.meganck@ugent.be.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Meganck
authors.affiliation: Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Faculty of Bioscience Engineering, Department of Biosystems, KU Leuven, Kasteelpark Arenberg 30, bus 2456, B-3001 Heverlee, Belgium.
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Goddeeris
authors.affiliation: Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Edith
authors.initials: E
authors.lastname: Stuyven
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Sofie
authors.initials: S
authors.lastname: Piepers
authors.affiliation: Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Cox
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Opsomer
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology; D002417:Cattle / Q000276:immunology / Q000502:physiology*; D049109:Cell Proliferation; D003126:Colostrum / Q000166:cytology* / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary*; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D008264:Macrophages / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology
keywords: Cattle; Cell proliferation; Colostrum; Flow cytometry; Leukocytes