Figure EV2. Flow velocity resolves into a normal force on the TCR/pMHC as a result of anchoring flexibility.

Biotinylated pMHC and antibody‐bound TCR have rotational freedom as a result of a flexible linker between the avi‐tag and the pMHC and between the TCR and the his‐tag that the antibody binds. This allows their binding site to diffuse in volumes represented by potions of disc in light blue for the pMHC and light red for TCR. Near to the surface of the chamber, the flow velocity is given by a first‐order approximation: v(z) = Gz, where z is the distance to the surface in nm and G is the shear rate constants in s⁻¹. The shear stress T_s (in N/m² or pascals) applied corresponds to the force applied by the flow per surface unit and is calculated by the product between G and the viscosity of the medium (μ) in Pa/s: T_s = μG. Taking into account the dimensions of the LFC section lxH, shear rate can be determined for a given flow Q using the formula: G ≈ 6Q/H². When the microsphere makes a link with the surface the flow creates a hydrodynamic force (R) given by the equation: R ≈ 32μa²G =1.7005 × 6πμa²G where a is the radius of the microsphere. The microsphere is also subjected to a torque force (Γ): Γ ≈ 0.9440 × 4μa³G. In addition, a lever effect increases the force applied to the interaction, so the total force applied (F) is given by the equation: F ≈ (R + Γ/a)√(a/2L) where L is the length of the formed bond, that is, the length of the link. Additional details can be found in (Pierres et al, 1995).