Extraction of Ex Vivo Macrophages from the Renal Medulla of Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is recognized as a versatile model for hematopoietic studies due to its transparency, genetic similarity to humans, and ease of manipulation. This study describes a protocol for the ex vivo extraction and differentiation of macrophages from the renal medulla of zebrafish using transgenic lines and L929 cell-conditioned medium (LCCM) as an alternative to recombinant M-CSF. Adult TgMpegmCherry zebrafish were maintained under controlled conditions. Following euthanasia, renal medullary cells were isolated, treated with antibiotics, and cultured in either LCCM or recombinant human M-CSF. Macrophage differentiation was assessed using confocal microscopy (mCherry), flow cytometry, and functional phagocytosis assays. The protocol enabled efficient differentiation of progenitor cells into macrophages, with an average of 49.1% mCherry+ cells after 7 days in LCCM, outperforming recombinant M-CSF. Differentiated cells demonstrated strong phagocytic activity, confirming macrophage functionality. This method provides an accessible approach to obtaining ex vivo zebrafish-derived macrophages, enabling immunological and hematopoietic studies and allowing for functional comparisons with traditional murine macrophage protocols.