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The toolbox for research in the equine immune system is rather limited. Accordingly attempts to use markers or molecules established in the human system is one approach to extend the options. Here, a first analysis was performed using selected antibodies not yet included in human leukocyte differentiation antigen workshop 8 (HLDA-8). Many antibodies of this study were directed against markers of the myelo-monocytic lineage of cells. A further aim of this study was to set up and standardize the conditions required for the equine part of HLDA-8.
66 mouse anti-human monoclonal antibodies from different companies were used. Most of them were directly conjugated and of those most were PE-labelled (a few FITC). The other antibodies were detected using a goat anti-mouse PE-labelled secondary antiserum. Equine leukocytes were isolated by Ficoll (1.090) gradient centrifugation. Equine PBMC were isolated by an additional Ficoll (1.077) gradient. Monocytes were isolated and differentiated as described. Analysis was performed using a FACSCalibur and multiwell auto sampler for screening. Acquisition and gating of cell subsets (granulocytes, monocytes, lymphocytes, platelets) was performed using two different settings (leukocytes vs. platelets) and the scattergramms according to protocols established earlier.
Antibodies with homogenous staining of cell populations according to knowledge in humans were considered specific and include antibodies against CD14, CD83, MHC class I and II. Second, antibodies that showed staining of populations, but did not match with knowledge in humans or showed staining of subpopulations. Those two groups may not be considered specific until further analysis such as immuno-precipitation is performed.