![]() Due to a number of limitations, afferent lymph vessels cannot be readily cannulated in mice or humans, and lymph vessel cannulation in sheep allows for the analysis of lymphocytes during their physiological recirculation through tissues. Our laboratory studies lymphocyte recirculation using lymph vessel cannulation in sheep, which was pioneered by Bede Morris. Due to this absolute and relative lack of reagents, the design of state-of-the-art multicolor flow cytometry staining panels is much more difficult than it is for mouse or human cell samples. In addition, antibodies for non-standard species tend to be more expensive. It is also not uncommon to receive limited amounts of hybridoma supernatant rather than purified antibody. Unfortunately, we face many limitations in the analysis of non-mouse animal samples, including lower availability of commercially or otherwise available antibodies to cell antigens and reduced options for fluorochrome labels by commercial antibody suppliers. įlow cytometry is a key method in immunological studies encompassing biomedical, veterinary, agricultural, and wildlife research, but the method is also routinely employed in veterinary clinical laboratories. More recent examples for the use of non-mouse species in biomedical research include pigs and sheep in orthopedics and Alzheimer’s disease and dogs in oncology. An example is the guinea pig, which has been a model for human infectious diseases for 200 years, and enabled disease research and vaccine development in tuberculosis. However, animal species other than the house mouse may represent more suitable models of specific human physiology, disease or anatomy, and can also enable studies of comparative medicine and/or of zoonotic pathogens in their natural hosts. The house mouse ( Mus musculus) is the most frequently used species in biomedical research and, as a consequence, a large spectrum of reagents and genetic models are available. Therefore, many institutions have acquired high capacity flow cytometers, and the analysis of > 10 fluorochromes has become routine in the study of human and mouse cells. Polychromatic experiments enable the simultaneous measurement of a larger number of cell surface and intracellular markers, thereby facilitating the analysis of infrequent cell subsets or limited cell samples. flow cytometry analyzers and sorters) allow for the theoretical analysis of up to 50 parameters in a single staining panel, and a 28-color panel has recently been demonstrated. Recent advances in the development of novel fluorochromes and instrumentation (i.e. Flow cytometry employs fluorochrome-labeled antibodies that detect cell surface or intracellular antigens, a method that was first developed for characterization of cells and tissues by microscopy. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study.įluorescence-activated cell sorting (FACS) and flow cytometry have been essential immunological tools since the invention of FACS in the late 1960s, as they enable identification, characterization, and isolation of defined leukocyte subsets. Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. ![]()
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