Abstract
The vertebrate immune system comprises both adaptive and innate immune cells with distinct functions during the resolution of inflammation and wound healing after tissue injury. Recent evidence implicates a requirement for innate immune cells from the myeloid lineage during the early stages of limb regeneration in the Mexican axolotl. Understanding the functions of innate and adaptive immune cells in the axolotl has been hampered by a lack of approaches to isolate and analyze these cells. Here we describe a protocol to isolate myeloid cells from the regenerating axolotl limb that incorporates intravenous delivery of physiological labels. In addition we provide a protocol to enrich for leukocytes in the peripheral blood. These protocols produce single-cell suspensions that can be analyzed using flow cytometry or sorted into specific subsets using fluorescent-activated cell sorting (FACS). FACS is a routine approach to sort cells based on their physical characteristics as well as their cell surface antigen repertoire. Isolated cell populations can then be analyzed in a wide range of downstream assays to facilitate a greater understanding of leukocyte biology in the axolotl.
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Acknowledgments
We thank Nadia Rosenthal and the members of her laboratory for the advice and helpful discussions. We thank members of the FlowCore facility at Monash University for performing FACS experiments. This work was supported by grants from Stem Cells Australia (Australian Research Council). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.
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Debuque, R.J., Godwin, J.W. (2015). Methods for Axolotl Blood Collection, Intravenous Injection, and Efficient Leukocyte Isolation from Peripheral Blood and the Regenerating Limb. In: Kumar, A., Simon, A. (eds) Salamanders in Regeneration Research. Methods in Molecular Biology, vol 1290. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2495-0_17
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DOI: https://doi.org/10.1007/978-1-4939-2495-0_17
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