Hq csc38 tipless cr au

For the test-probe
calibration method,^{39 (link)} the normal and torsional
resonance frequency and quality factor of a UV–ozone-cleaned
cantilever (HQ/CSC38/tipless/Cr–Au, MikroMasch, Bulgaria) were
determined with an atomic force microscope (Nanowizard III, JPK, Germany)
to calculate the normal and torsional spring constants.^{40 (link)} Glass particles (50–100 μm, Whitehouse
Scientific, UK) were deposited on a microscope slide, dried with a
nitrogen jet, and mounted on the AFM stage. The tip of the cantilever
was dipped into a small drop of UV-curable adhesive (Norland Optical
Adhesive 63, USA) and aligned with a particle. The cantilever was
brought into contact with the particle and the adhesive was cured
with UV light (GEM10 UV, 3000 mW at 365 nm, Nitecore, Germany) for
1 min at a distance of 3–4 cm. A clean and sharp edge of the
silicon wafer was used as a vertical sidewall for the measurement
of lateral deflection sensitivity.

hCD4+ T cells were plated on a glass-bottom dish (Willco Wells) precoated with either ICAM1 (Life Technologies) or 0.01% poly-l-lysine (Sigma-Aldrich) and immersed in culture media solution (Life Technologies). Force spectroscopy AFM experiments were performed using a Bruker Bioscope Catalyst AFM system (Bruker) mounted on an inverted Axiovert 200 M microscope (Zeiss) equipped with a confocal laser-scanning microscope 510 Meta (Zeiss) and a ×40 objective lens (0.95 NA, Plan-Apochromat, Zeiss). The hybrid microscope instrument was placed on an acoustic isolation table (Kinetic Systems). During AFM experiments, T cells were maintained at a physiologically relevant temperature 37 °C using a heated stage (Bruker). A soft silicon nitride tipless AFM probe (HQ:CSC38/tipless/Cr-Au, MikroMasch) was used for T cell’s compression. The AFM microcantilevers were pre-calibrated using the standard thermal noise fluctuations method. The estimated spring constants for microcantilevers used were 0.07–0.1 N/m. After calibration, the AFM probe was moved on top of a rounded T cell. Five to ten successive force curves were performed on each T cell. The deflection set-point was set to 20 nm yielding applied forces between 1.5 and 2 nN.