Mechanical challenge of human RBCs by atomic force spectroscopy. (A) Schematic drawing of the mechanical challenge owing to application of the cantilever of an AFM with forces of up to 400 pN applied. RBCs were attached to the cantilever with Cell-Tak (BD Biosciences, San Jose, CA) and spontaneously adhered to the microscope dish (microdish). (B) Schematic drawing showing the retraction of the cantilever with measurement of the separation force. (C) Confocal image of RBCs labeled with the membrane dye CellMask (Life Technologies, Carlsbad, CA) at 1:2000 for 10 minutes. The yellow arrow points to a tether, clearly connecting 2 RBCs. Scale bar, 10 μM. (D) Two RBCs were pushed together and separated, as outlined in panels A and B, in a plasma-like solution containing 40 mg/mL dextran (70 kDa). Shown are optical sections and the 3D reconstruction with clearly visible tethers in the x/z and y/z planes (yellow arrows) but few visible tethers in the x/y plane. The 3D reconstruction, in addition to the optical sections, reveals uneven staining of the tethers, suggesting the presence of lipid rafts or other molecular agglomerations. It is of little importance whether the tethers form between RBCs or between an RBC and the substrate, as protein transfer could potentially also occur between RBCs and structures of the spleen. (E) Even without the formation of tethers, RBCs strongly interact, potentially allowing a protein transfer. (F) Atomic force spectroscopy measurement reflecting the situation imaged in panel E with an RBC interaction time of several seconds. (G) Representative atomic force spectroscopy measurement with a contact time close to 0 and a velocity of the cantilever movement of 5 μm/s in a dextran solution (40 mg/mL dextran 70 kDa) used to mimic plasma. Such a probing is too fast to be imaged by confocal microscopy.