High‐throughput cell mechanical phenotyping for label‐free titration assays of cytoskeletal modifications

Abstract

The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label‐free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real‐time deformability cytometry (RT‐DC), a novel microfluidic tool to examine the effects of biochemically modified F‐actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half‐maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F‐actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT‐DC. Drug‐induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high‐throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label‐free dose‐response assays of cytoskeletal modifications.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage

“…The observation of cell stiffening during differentiation in DC could, therefore, be attributed to the previously described phenomenon of increased nuclear stiffness in differentiated cells (Pajerowski et al, 2007). RT-DC, in turn, deforms cells within a few milliseconds with sub-µN forces, and was recently shown to be primarily sensitive to cytoskeletal perturbations, in particular to those related to the actin cytoskeleton (Golfier et al, 2017).…”

Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing

“…The dataset and methods for cell preparation have been published earlier 30 . Briefly, the human peripheral promyelocytic leukemia cell line HL-60 was cultured in RPMI-1640 (Life Technologies, Carlsbad, USA) + 10% fetal bovine serum and 1% penicillin/streptavidin (Sigma-Aldrich, St. Louis, USA) at 37 °C and 5% CO 2 .…”

“…In contrast to DD , the relative deformation RD introduced earlier relates the observed difference in one sample, e.g., control measurement M i , to another sample, e.g., treatment measurement M j by: $RD=\frac{M_{j,\text{Ch}}-M_{j,\text{Res}}}{M_{i,\text{Ch}}-M_{i,\text{Res}}}.$ 30 This parameter RD reduces the discrimination of two samples to a single number rendering statistical analysis challenging.…”

Microfluidic deformability-activated sorting of single particles

“…Traditional single-particle deformability measurements include micropipette aspiration 23 , optical stretching 24 , atomic force microscopy (AFM) 1 , and magnetic bead-based rheology 2 . To increase the throughput, microfluidic approaches have been developed that rely on either the physical constriction 10,[25][26][27][28] or the hydrodynamic shear stress from a channel [29][30][31] , a cross-section 32 , or a Tjunction 33 .…”