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Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies
Vanessa Meganck1, Bruno M Goddeeris2, Edith Stuyven3
1Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Insights
This study presents a validated method for isolating bovine colostrum mononuclear cells (CMC) for immune function analysis. The findings confirm CMC viability and responsiveness, crucial for understanding passive immunity in calves.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Neonatal Immunology
Background:
- Bovine colostrum is a vital source of passive immunity for newborns.
- Colostrum contains leukocytes (CMC) that may play a significant role in immune development.
- Existing isolation protocols for bovine CMC lack validation and comprehensive phenotypic analysis.
Purpose of the Study:
- To develop and validate a method for isolating bovine colostrum mononuclear cells (CMC).
- To characterize the phenotype of isolated CMC using flow cytometry.
- To assess the functional capacity (proliferative response) of isolated CMC.
Main Methods:
- Isolation of CMC from bovine colostrum using density gradient centrifugation.
- Phenotypic analysis by flow cytometry with specific bovine cell surface markers.
- Assessment of cellular proliferation using a (3H)-thymidine incorporation assay with mitogen stimulation.
Main Results:
- A reproducible method for CMC isolation was established, yielding sufficient cell counts for analysis.
- Flow cytometry identified T lymphocytes (25.4%), B lymphocytes (2.9%), and macrophages (32.7%) within CMC.
- Isolated CMC demonstrated viability and proliferative capacity upon stimulation with concanavalin A.
Conclusions:
- The developed method provides a reliable means for isolating and characterizing bovine CMC.
- Phenotypic and functional analysis of CMC is feasible and repeatable.
- These findings support the importance of CMC in bovine passive immunity and provide a foundation for further research.
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.
