Atomic force microscopy stiffness measurements

Live cells were probed utilizing an MFP-3D-BIO atomic force microscope (Asylum Research) and a DNP cantilever (Bruker) with nominal spring constant 0.06 N/m. The cantilever was calibrated before each measurement to ensure accuracy. Cells were seeded on 60 mm poly-d-lysine–coated Petri dishes using DMEM (Life Technologies) with 10% heat-inactivated horse serum (Life Technologies), 5% fetal bovine serum (Atlanta Biologicals), and 1% penicillin/streptomycin. After 24 h of recovery, cells were differentiated using media containing DMEM, 1% heat-inactivated horse serum, and 1% penicillin/streptomycin. Added to this was a 1:1000 ratio of 100 ng/µl nerve growth factor (NGF) (Novoprotein). This was added to the cells a minimum of 24 h before measurements were taken.

Cells were viewed 2 d after plating and 1 d after treatment with nerve growth factor (NGF). Cells with minimal cell–cell contact were selected to reduce the mechanical impacts of cell communication. Three force curves in separate perinuclear regions with cantilever velocity of 2 µm/s and trigger point lnN were taken for each selected cell. Measurements were taken within 30 min of removal from the incubator to facilitate cell viability.

The stiffness of the measured cells E was determined from the force curve data utilizing the Hertz model for conical cantilever tip geometry,

where k is the cantilever spring constant, d is the cantilever deflection, ν is the Poisson’s ratio (0.5 used for an assumed incompressible material), ∆ is the sample indentation depth, and φ is the half­-angle of the conical cantilever tip (35°). The force curves were processed using a custom MATLAB code that fits the indentation curve over a 500 nm range after manual selection of the initial contact point.