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Updated: Aug 8, 2026
Isolation and Activation of Murine Lymphocytes
Published on: October 30, 2016
Isolation, characterization and in vitro mitogenic stimulation of peripheral blood lymphocytes from an American
1Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon.
Insights
Researchers developed a fast method to isolate bison leukocytes. This technique yields pure, viable immune cells, including T lymphocytes, for further study.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Mammalian Hematology
Background:
- Leukocyte isolation from American buffalo (bison) peripheral blood is crucial for immunological research.
- Existing methods may lack efficiency or reproducibility for bison samples.
Purpose of the Study:
- To establish a rapid and reproducible method for isolating and characterizing leukocytes from American buffalo.
- To assess the purity, viability, and functional activity of isolated bison leukocytes.
Main Methods:
- Peripheral blood buffy coat cells were isolated using Percoll gradient centrifugation (1.079 g/mL).
- Sheep erythrocyte-rosetting technique was employed to identify T lymphocytes (ER+).
- Flow cytometry with Fluorescein isothiocyanate (FITC)-conjugated lectins (peanut agglutinin, concanavalin A) was used for cell characterization.
Main Results:
- High yields of pure, viable leukocytes were obtained via Percoll gradient centrifugation.
- Sheep erythrocyte-rosetting identified 59% T lymphocytes.
- FITC-lectin staining indicated high positivity for peanut agglutinin (77%) and concanavalin A (89%).
- Isolated leukocytes demonstrated functional activity, including proliferation in response to mitogens and recombinant bovine interleukin-2.
Conclusions:
- A robust method for bison leukocyte isolation and characterization has been established.
- The isolated leukocytes are viable, pure, and contain functionally active T and B lymphocytes.
- This method supports further immunological investigations in American buffalo.
Abstract:
A rapid and reproducible method is described for the isolation and characterization of leukocytes from the peripheral blood of an American buffalo (bison). Centrifugation of the buffy coat cells on a Percoll gradient (1.079 g/mL) at 650 x g for 20 min resulted in the separation and high yields of pure viable leukocytes. The sheep erythrocyte-rosetting technique (ER) showed that 59% of the cells were ER+ (T lymphocytes). Fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin and FITC-conjugated concanavalin A revealed 77% and 89% positive cells, respectively. The isolated leukocytes contained adherent accessory cells and functionally active T and B lymphocytes which proliferated in response to both T and B cell mitogens and to exogenous recombinant bovine interleukin-2 in the absence and/or presence of the thiol compound 2-mercaptoethanol.
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