We adapted an original technique with the help of E. Evans Lab (UBC, Vancouver), namely the Biomembrane Force Probe (BFP), based on micropipette manipulations and able to measure forces down to a few pN. This technique uses a RBC as a spring on which a bead is glued and used as the probe. Our homemade version is able to perform classical force ramps, adhesion frequency analysis and force clamp measurements
Biomembrane force probe in bead/bead and bead/cell configurations. The aspirated streptavidinated RBC serves as an extrasoft and tunable spring. The bead that is glued at its apex serves as the probe surface and is decorated with biotin and the desired hapten. This bead is tracked at 1000fps, and the target surface, either a bead or a cell under a controlled tension (using a delicate aspiration) is moved at controlled speed using a piezo device and contact is made under a controlled force for a given time. The deformation of the RBC, as monitored via the motion of the probe bead, allows to gain information, similarly to the AFM in force mode, on elasticity (in the case of cell) or adhesion at the single molecule level. Bead is ~ 2µm in diameter
We compared this BFP in terms of stability, capability to reach single molecule resolution, force resolution to AFM with classical adhesion systems (streptavidin / biotin), and tested it on TCR/pMHC systems at T cell surface.
This set-up is continuously developed and we plan to adapt it as a Lateral AFM following with the help of Heinrich lab (UC Davis).
This works was supported by CNRS Prise de risque, HFSP grant, ANR JCJC DissecTion, CNRS PEPS grants to PH Puech.
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