Microfluidic adhesion experiments and reflection interference contrast microscopy

Microfluidic experiments were performed as previously described^22,25 using the air pressure-driven microfluidic system BioFlux 200 (Fluxion Biosciences, South San Francisco, CA). Channels of a 48-well BioFlux plate were biofunctionalized for 3 h at 37 °C with either 2% BSA serving as a negative control or plasmatic VWF (100 µg/ml) and rinsed with 0.5% BSA. Channels were perfused for 10 min with different erythrocyte or artificial vesicle preparations at 2 dyne/cm² and 10 dyne/cm², respectively. Reflection interference contrast microscopy (RICM) was used to visualize the interactions of the erythrocytes with the coated channel surfaces.

For RICM visualization, an inverted Microscope Observer Z1 (Zeiss, Jena, Germany) with a Colibri LED illumination system was used at two excitation wavelengths (470 and 555 nm). Images were captured with a monochromatic camera (Axiocam MRm, Zeiss, Jena, Germany). The acquisition time was 180 ms at 200-ms or 350-ms intervals. For image acquisition and processing, we used ZEN software (Zeiss, Jena, Germany). The computerized quantification of erythrocyte-VWF adhesion events was performed using an adaptive Hough transformation model²⁶.