Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.
Abstract: CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. Objective: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. Methods: Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). Results: Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2-fold higher than in previous published outcomes. Conclusions: The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. Conclusions: The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.
© 2012 EVJ Ltd.
Publication Date: 2012-09-19 PubMed ID: 22994596DOI: 10.1111/j.2042-3306.2012.00616.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study details an improved protocol to enhance Dendritic Cell (DC) generation in horses from CD14+ cells, using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. The technique increases the quantity of these cells, which play a crucial role in immune responses and therapies.
Objective and Methodology
- The aim of this study was to establish a protocol to enhance the generation of dendritic cells (DCs) from CD14+ cells in horses. This was to overcome the current limitations in horse therapy, as the efficiency of generating DCs from adherent peripheral blood mononuclear cells (PBMCs) is relatively poor.
- CD14+ PBMCs were isolated from fresh blood samples of 3 horses. The cells were stained with a mAb against human CD14 and a secondary phycoerythrin (PE) conjugated antibody to determine the initial concentration of CD14+ cells in the sample.
- These cells were sorted using an automated magnetic activated cell sorting (MACS) system using the same primary and secondary antibodies.
- The remaining CD14+ cells were cultured for 4 days with additional granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the culture media. The generation of DCs was determined by morphological analysis and the expression of surface marker proteins (hCD83, hCD86, eqMHCII).
Findings
- The results of the study demonstrate that prior to selection, CD14+ cells were 5.5% of the total cell population. After sorting and selecting for CD14+ cells, the percentage increased to 78.46%.
- After positive cell selection, 98% of the population were CD14+ cells and the cell viability was not affected by the protocol. The resulting DCs exhibited the desired morphology and surface marker proteins.
- The generated DC yield, upon culture, was twice as much as in previous published studies. This indicates that the new protocol resulted in a significant increase in the production of DCs from CD14+ cells in horses.
Conclusion
- Overall, the implementation of an additional CD14 cell separation step after PBMC isolation significantly increased the number of CD14+ cells, thus, enhancing the number of generated DCs. This advancement will be instrumental in improving therapeutic approaches involving these cells in horses.
Cite This Article
APA
Durán MC, Willenbrock S, Carlson R, Feige K, Nolte I, Murua Escobar H.
(2012).
Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting.
Equine Vet J, 45(2), 249-253.
https://doi.org/10.1111/j.2042-3306.2012.00616.x Publication
Researcher Affiliations
- Equine Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
MeSH Terms
- Animals
- Antibodies, Monoclonal
- Automation
- Cell Separation / methods
- Cell Separation / veterinary
- Cell Survival
- Gene Expression Regulation / physiology
- Horses / blood
- Humans
- Lipopolysaccharide Receptors / genetics
- Lipopolysaccharide Receptors / metabolism
Citations
This article has been cited 3 times.- Bowlby CM, Purmessur D, Durgam SS. Equine peripheral blood CD14(+) monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation. Vet Immunol Immunopathol 2023 Jan;255:110534.
- Rzepecka A, Żmigrodzka M, Witkowska-Piłaszewicz O, Cywińska A, Winnicka A. CD4 and MHCII phenotypic variability of peripheral blood monocytes in dogs. PLoS One 2019;14(7):e0219214.
- Spiesschaert B, Goldenbogen B, Taferner S, Schade M, Mahmoud M, Klipp E, Osterrieder N, Azab W. Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a Dynamic In Vitro Model. J Virol 2015 Dec;89(23):11899-908.
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