High-throughput analysis of membrane fluidity unveils a hidden dimension in immune cell states

Cell membranes undergo biophysical remodelling as an adaptation to the surroundings and to perform specific biological functions. However, the extent and relevance of such changes in human immune systems remain unknown, largely due to the lack of high throughput and multidimensional methodologies. Here, we describe a cytometry-based method with single-cell resolution which fills this technological gap by combining biophysical profiling with conventional biomarker analysis. This platform allows to reveal notable cell type-dependent remodelling of membrane fluidity during immune stimulations and in diseases. Using immune cells exposed to tumour microenvironment as well as from long COVID and chronic lymphocyte leukaemia patients, we demonstrate that membrane fluidity is orthogonal to surface marker expression. Moreover, this biophysical parameter identifies new functional and pathological states of immune cells previously undetected via surface marker profiling alone. Our findings will contribute to a more precise definition of immune cell states based on their biophysical properties and will pave the way for a better understanding of the functional heterogeneity of immune cells. ### Competing Interest Statement The authors have declared no competing interest.