Related Experiment Video
Updated: Aug 11, 2026
Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation
Published on: January 26, 2016
The effect of different isolation procedures on canine leucocyte populations and on lectin-induced lymphocyte
T de Bruin1, H de Rooster, H van Bree
1Department of Diagnostic Imaging of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. tanya.debruin@ugent.be
Insights
Optimizing canine lymphocyte isolation for immunology research is crucial. A novel method combining carbonyl iron/arabic gum treatment, density gradient centrifugation, and adherence to fibrinogen-coated flasks significantly improves purity and yield, preserving cell proliferation capacity.
Area of Science:
- Veterinary Immunology
- Cell Biology
- Immunological Assays
Background:
- Peripheral blood mononuclear cells (PBMC) are vital for immunology research.
- Contaminating cells can affect lymphocyte proliferation assays.
- Canine lymphocyte purification presents challenges in purity and yield.
Purpose of the Study:
- To optimize canine lymphocyte isolation for purity and yield.
- To ensure isolated cells retain proliferation capacity.
- To evaluate different density gradient media and pre-treatment methods.
Main Methods:
- Examined various density gradient media for canine lymphocyte isolation.
- Investigated pre-treatment with carbonyl iron/arabic gum for phagocyte removal.
- Assessed adherence to fibrinogen-coated flasks.
- Measured cell activity via lectin-induced proliferation.
Main Results:
- A combined method (carbonyl iron/arabic gum, density gradient medium 'G', fibrinogen-coated flasks) yielded optimal results.
- Achieved median lymphocyte purity of 88% and median yield of 54%.
- The optimized method maintained high cell proliferation and stimulation index.
Conclusions:
- The developed protocol significantly enhances canine PBMC isolation.
- This method provides high-purity lymphocytes with preserved proliferative function.
- It is suitable for experimental and clinical immunology studies in canines.
Abstract:
Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.
