PhD Scientific Days 2021

MO_II_L: Molecular Sciences II. Lectures

Monitoring Osteoclast Development with a Fluorescence-based Method

Előadó neve

Mr. Pánczél, Áron

Előadó munkahelye

Semmelweis University, Faculty of Medicine, Department of Physiology

Előadó telefonszáma

0036303461067

Előadó e-mail címe

panczel.aron@med.semmelweis-univ.hu

Az előadás címe

Monitoring Osteoclast Development with a Fluorescence-based Method

Szerző(k) neve és munkahelye

Áron Pánczél1, Attila Mócsai1
1 Semmelweis University, Faculty of Medicine, Department of Physiology

Bemutatás módja

Szóbeli

Szekció

Molecular Sciences II. Lectures

Language of the presentation

English

Preferred session

Molecular Sciences

Összefoglaló szövege

Introduction • Osteoclasts are multinucleated syncytia of myeloid origin responsible for bone resorption under various physiological and pathological conditions such as osteoporosis, bone-metastasis or autoimmune arthritis. Though they can be readily differentiated in vitro, the quantitative evaluation of such experiments is at present rather challenging.
Aims • The objective of our study was to develop fluorescence-based methods for efficient, real-time monitoring of the development of osteoclast cultures.
Methods • Our osteoclastogenesis assays are based on two genetic modifications in mice. The cathepsin K cre (Ctskcre) mutation results in the osteoclast-specific expression of the cre recombinase, while the mTmG mutation is a cre-responsive reporter switching from red to green fluorescence upon recombination.
Results • First we crossed Ctskcre and mTmG-carrying mice to obtain a Ctsk-reporter strain. This resulted in robust green signals in osteoclast cultures. Green fluorescence emerged in both mononuclear pre-osteoclasts and multinuclear osteoclasts. Then, we set up a second assay where Ctskcre cells were co-cultured with mTmG cells as a fusion-specific reporter. Here only upon cell-to-cell fusion could cre access its mTmG substrate and thus activate green fluorescence. This time a less robust green fluorescence could be detected that seemed to be confined to multinuclear syncytia but not to mononuclear cells. Parallel macrophage cultures, as well as osteoclast cultures lacking either the Ctskcre or the mTmG mutation, were devoid of green fluorescence indicating the osteoclast-specific nature and confirming the genetic working principle of both assays. We also demonstrated the underlying mechanics of our assays at genomic DNA, transcriptional and protein levels.
Conclusion • In summary, we successfully established two fluorescence based assays that enabled us to follow real-time changes in cell cultures differentiated towards osteoclasts. The included control cultures confirm a high degree of specificity for osteoclasts while the possibility of automated quantification highlights the potential of our approach for high-throughput (e.g. drug-screening) scenarios.
Funding • This work was supported by the Hungarian National Scientific Research Fund (NKFIH-OTKA No. K119653 and NVKP_16-1-2016-0039).

University and Doctoral School

Semmelweis University, Doctoral School of Molecular Medicine

Supervisor

Prof. Dr. Attila Mócsai

Publication of my abstract

I give consent to the publication of my abstract on the website of the congress.

Kind

Szabad

Status

elfogadva

Accepted presentation method

szóbeli

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

5996

Start

12:35

End

12:50

Authors (legacy)

Áron Pánczél1, Attila Mócsai1
1 Semmelweis University, Faculty of Medicine, Department of Physiology