Neutrophil microrheology probed by Atomic Force Microscopy

Neutrophil rheology and the changes it suffers following activation play an important role in neutrophil arrest and extravasation during passage through microcapillaries. However, the magnitude and frequency dependence of neutrophil stiffness remains poorly defined. Atomic Force Microscopy (AFM) is suited for measuring neutrophil rheology with minimal cell perturbation. The aim of this work was to measure the complex shear modulus (G*=G’+iG’’) of resting and activated neutrophils with AFM. G* was measured with spherical AFM tips (r = 2.25 μm) over 3 frequency decades (0.1–100 Hz) for neutrophils isolated from 3 Sprague‐Dawley rats plated on uncoated glass slides (activated state, n=16) or coated with poly(HEMA) (resting state, n=15). G* was calculated from AFM force‐indentation relationships by applying a multifrequency signal of 50 nm amplitude around an indentation setpoint of 0.5 μm. The Hertz contact model between two spheres (cantilever tip and neutrophil) was used. G’ and G’’ at 1.6 Hz were of 234 ± 66 Pa (mean ± SEM) and 58 ± 17 Pa respectively for resting neutrophils. Both G’ and G’’ followed a power‐law behavior with frequency with an exponent α=0.16. Activated neutrophils had G’ = 862 ± 238 Pa and G’’= 176 ± 37 Pa and also followed a power‐law behavior with α=0.13. AFM allowed us to measure the absolute magnitude of neutrophil stiffness. Both the elastic (G’) and loss (G’’) moduli followed a scale‐free positive frequency dependence. Activated neutrophils had a stiffness 4‐fold higher and a smaller power law exponent. This behavior is consistent with soft glassy rheology. This work was supported by NAN2004‐09348‐C04‐04