Jpk nanowizard 1

Cells were seeded and treated on fluorodishes. Cell compliance measurements were conducted on a JPK Nanowizard-1 (JPK Instruments, Berlin, Germany) operating in force spectroscopy mode, mounted on an inverted optical microscope (IX-81; Olympus, Shinjuku-ku, Japan). AFM pyramidal cantilevers (MLC-2T; Bruker, Billerica, MA, USA) with a spring constant of 0.03 N/m were used with a 15 μm diameter glass bead attached to cantilever tip. Prior to measurements with the adapted cantilevers, their sensitivity was calculated by measuring the slope of force-distance curve in the AFM software on an empty region of the petri dish. For indentation tests, the cantilever was aligned over the cell away from the nucleus, and for each dish, 30 force curves were acquired across 30 cells. Force-curve acquisition was carried out with an approach speed of 5 μm/s and a maximum set force of 1 nN. Elastic moduli were calculated from the force-distance curves by fitting the contact region of the approach curve with the Hertz contact model [70 (link)] using the AFM software (JPK).

For AFM study, cell seeded
polyacrylamide gels on coverslips were lifted from 24-well plates
prior to measurement and immediately attached to a Petri dish with
a droplet of cyanoacrylate adhesive, applied with a 10 μL pipet
tip. After coverslip attachment (∼1 min), 100 μL of culture
medium (DMEM 10% FBS) was applied to the coverslip in order for the
AFM measurements of cells to be conducted as soon as possible (<1
h). Measurements of HSCs on polyacrylamide gels were conducted on
a JPK Nanowizard-1 (JPK instruments) AFM operating in force spectroscopy
mode, mounted upon an inverted optical microscope (IX-81, Olympus).
AFM pyramidal cantilevers (MLCT, Bruker) with a spring constant of
0.03 N/m were used. Before conducting measurements, cantilever sensitivity
was calculated by measuring the force–distance slope in the
AFM software on an empty Petri dish region. For cell indentation measurements
the cantilever was aligned over a central region of a cell using a
20× objective and the optical microscope. For each cell 3–5
force curves were acquired at an approach speed of 2 μm/s and
a maximum set point of 0.1 V to ensure that the cantilever only probed
the cell membrane. The force–distance curves were used to calculate
elastic moduli in the AFM software through the application of the
Hertz contact model.^{93 (link)}