Fluorescence-Based Real-Time Analysis of Osteoclast Development

• DOI: 10.3389/fcell.2021.657935
• Publication Date: 2021-07-13T07:50:22Z
• Authors: Áron Pánczél; Simon P. Nagy; János Farkas; Zoltán Jakus; Dávid S. Győri; Attila Mócsai
• Keywords: Cathepsin K; Cell fusion

Osteoclasts are multinucleated cells of hematopoietic origin which critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes intercellular fusion. This process is directed by M-CSF RANKL also able to trigger osteoclast development marrow vitro . conventionally visualized histochemical staining followed manual counting, hinders kinetic studies automated quantification. Here we describe two fluorescence-based assays for the real-time analysis cell (FRAMCO) primary mouse cultures. Both rely on red-to-green fluorescence conversion membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene Cre recombinase driven osteoclast-specific cathepsin K promoter (Ctsk-Cre). In first assay (FRAMCO1.1), triggers color carrying both Ctsk-Cre mTmG transgenes. second (FRAMCO1.2), triggered fusion neighboring co-cultured separately either or The were tested using a high-content confocal imaging system, FRAMCO1.1 showed robust more than 50% culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. FRAMCO1.2 less but still readily measurable multinuclear 5 differentiation. required transgenes gave no signals parallel macrophage proper functioning was confirmed at DNA, mRNA bulk protein level. systems validated lisophosphatidylcholine, previously reported inhibitor preosteoclast Taken together, our allow high-throughput critical aspects development, facilitating screening novel drug candidates pharmacological control osteoclast-mediated