PhD Scientific Days 2025

Poster Session II. - G: Pharmaceutical Sciences and Health Technologies

Lineage-Specific Analysis of Basal Tyrosine Phosphorylation in Circulating Leukocytes of Hck–/–Fgr–/–Lyn–/– Mice

Name of the presenter

Deli Dorottya

Institute/workplace of the presenter

Department of Physiology, Semmelweis University, Budapest, Hungary

Authors

Dorottya Deli¹, Dorottya Markó¹, Krisztina Futosi¹, Attila Mócsai¹

1: Department of Physiology, Semmelweis University, Budapest, Hungary

Text of the abstract

Introduction: Tyrosine kinases are central regulators of immune cell signaling and are key drug targets in inflammatory diseases. We recently developed a rapid, quantitative flow cytometry–based assay to assess basal tyrosine phosphorylation of circulating neutrophils in mice.

Aims: We aimed to extend this method to allow parallel analysis of multiple leukocyte subsets and enable cell lineage–specific resolution in complex in vivo models.

Methods: Peripheral blood was collected from wild type and Hck–/–Fgr–/–Lyn–/– triple knockout mice, as well as from mixed bone marrow chimeras generated using knockout donor cells. Samples were stained with fluorescent antibodies specific for neutrophils, monocytes, B- or T-cells. After fixation and permeabilization, tyrosine-phosphorylated proteins were detected intracellularly using fluorescently labeled anti-phosphotyrosine or isotype control antibodies. For specificity control, anti-phosphotyrosine antibodies were preincubated with 1 mM soluble phosphotyrosine. Fluorescence signals reflecting basal phosphorylation were analyzed by flow cytometry.

Results: The assay revealed high basal tyrosine phosphorylation in all leukocyte populations. In Hck–/–Fgr–/–Lyn–/– mice, myeloid cells showed a marked reduction in phosphorylation, while lymphocytes remained unaffected. In mixed bone marrow chimeras, knockout and wild-type myeloid cells were distinguishable based on differential phosphorylation, demonstrating the method’s suitability for lineage-specific in vivo analysis.

Conclusions: This optimized assay enables rapid, quantitative, cell-type–resolved analysis of basal tyrosine phosphorylation in vivo. Its use in mixed chimeras allows within-animal comparison of wild-type and kinase-deficient myeloid cells, supporting its utility in lineage-specific functional studies and translational signaling research.

Funding: Funded by the Hungarian National Research, Development and Innovation Office (KKP-129954, FK-146729 and TKP2021-EGA-24) and the HUN-REN Hungarian Research Network (0207007)