Recombinant Protein-Coated Microspheres and RBCs

The recombinant protein-coated microspheres and red blood cells (RBCs) were prepared according to previously published methods.^{60 (link)–64 (link)} Briefly, for force-clamp assay, borosilicate glass microspheres (Cat. #9002, Thermo Fisher) were first chemically modified with -SH group through 3-mercapto-propyl-trimethoxysilane (Cat. #175617, Sigma-Aldrich), then incubated with streptavidin-maleimide (Cat. #S9415, Sigma-Aldrich) overnight in 200 mM phosphate buffer (pH = 6.7) at room temperature (RT). For the adhesion frequency assay, human RBCs were directly reacted with biotin-PEG3500-NHS (Cat. #62717, JenKem, China) at RT for 30 min in 10 mM HEPES buffer (containing with 145 mM NaCl, roughly 300 mOsm osmotic pressure, pH = 7.4) and then coated by streptavidin in HEPES buffer containing 1% BSA at RT for 30 min. Finally, suitable biotin-labeled recombinant protein was coated on microspheres or RBCs through biotin-streptavidin reaction in HEPES buffer containing 1% BSA at RT for 30 min.