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Supplementary MaterialsSupplementary Information 41467_2019_8370_MOESM1_ESM

Supplementary MaterialsSupplementary Information 41467_2019_8370_MOESM1_ESM. of mechanised cell assays to fast on-the-fly phenotyping of large sample sizes, but has been restricted to single material parameters as the Youngs modulus. Here, we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition, our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters Pentagastrin independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties. Introduction With the potential for label-free phenotyping of cellular states and functions, the mechanical properties of cells have gained an increasing importance over Rabbit Polyclonal to ACHE the last years1C3. Being sensitive to cytoskeletal and nuclear alterations, this biomarker has been used to track the stability, passaging, and differentiation of stem cells, to follow the activation of immune cells, and to characterize metabolic states4C8. As mechanical phenotyping is based on intrinsic cell material properties, it serves as a complementary approach to traditional molecular biology methods and is of an increasing importance in fundamental and applied research, where molecular markers are not wanted or not available. However, a broad translation of mechanical phenotyping into life science applications had so far been hampered by lack of a fast and robust measurement technique. While traditional methods like atomic force microscopy, micropipette aspiration, and optical stretching were limited to analysis rates of less than 100 cells per hour9C11, the introduction of microfluidic concepts increased the throughput by several orders of magnitude12,13. The serial deformation of cells in a hydrodynamic environment allows for throughput rates on the order of 100C10,000 cells per second, which is a prerequisite for screening applications, e.g., the combination of biophysical and molecular analysis or the characterization of highly potent skeletal stem cells in regenerative medicine14,15. In contrast to well established cell biology techniques, like flow cytometry, the parameter space of mechanical cell characterization cannot be extended by additional molecular markers simply, but is bound to any provided details that may be extracted from acoustical, mechanised, or optical measurements16C18. Nevertheless, cells are a long way away from a thermal equilibrium. Their response for an exterior mechanised load by means of creep or tension relaxation is extremely nonlinear and powered by both, a dynamic and a unaggressive intrinsic remodeling, which includes to become explored to web page link cytoskeletal properties to cell function19C21. While rheological tests and the perseverance of the frequency-dependent complicated modulus have primarily been performed on adherent cells2,22, microfluidic systems in conjunction with high-speed video microscopy allowed a rise in throughput and an expansion to suspended cells23,24. Utilizing a parallel selection of micron-sized constrictions, Lange et al. make use of the confinement of suspended cells within a microfluidic route to estimation cell fluidity and elasticity from movement swiftness, residence period, and generating pressure. Power-law rheology points out the collapsing of data from multiple cell lines and under multiple circumstances onto a get good at curve and it is in contract with the idea of gentle glassy components25,26. Quantitative deformability cytometry Pentagastrin expands this idea by presenting calibrated microspheres to remove quantitative details and permits potential evaluation Pentagastrin to reference strategies like micropipette aspiration27. As opposed to micro-constrictions, strategies like deformability cytometry (DC), real-time deformability cytometry (RT-DC) and real-time Pentagastrin fluorescence and deformability cytometry (RT-FDC) are contactless and make use of solely hydrodynamic tension to deform cells24,28,29. Furthermore, RT-FDC and RT-DC have the capability to execute picture acquisition and evaluation on-the-fly, which allows to get a label-free testing of heterogeneous cell examples of practically unlimited size as well as Pentagastrin the id of sub-populations predicated on mechanised properties. Nevertheless, in real-time data evaluation, picture acquisition and data evaluation have already been limited to an individual snapshot per cell and, thus only steady-state material parameters as the Youngs modulus can be derived30,31. Here, we introduce dynamic RT-DC (dRT-DC) for single cell rheological measurements in heterogeneous samples where we capture the full dynamics of suspended cells passing the central constriction of a microfluidic channel on-the-fly. We show that Fourier analysis of cellular shape modes allows to disentangle the complex cell response to time-dependent and time-independent hydrodynamic stress distributions, which are typical for almost any microfluidic system. The symmetry of the Fourier modes can be used to extract the stress-strain relationship and to determine viscoelastic cell parameters directly by applying simplest model assumptions. We show that our approach is impartial of cellular shape. Using a cell line as well as primary blood cells, we demonstrate that dynamic RT-DC is capable to determine an apparent Youngs modulus as well as an apparent viscosity with throughput rates of up to 100 cells per second. Interestingly, this technology allows for a rheological comparison amongst cells in a single measurement of entire blood. Furthermore,.