Microfluidic experimental setup for adhesion and recovery measurements of red blood cells in sickle cell disease.

Current microfluidic assays, which aim at quantifying mechanical properties of sickle cell red blood cells (SS-RBCs), suffer from a number of drawbacks in functionalization and flow control. Specifically, physical adsorption functionalization techniques produce inconsistent functional surfaces, and common volumetric flow pumps cannot be used to adjust the flow inside microchannels with minimal delay. We have designed an experimental setup that alleviates these complications by implementing aspiration for microchannel assembly that enables the use of most functionalization techniques and a pressure controller that allows instant and precise changes in the microchannel flow. Utilizing this setup, we have quantified SS-RBC adhesion to the integrin αvß3, a specific adhesion protein expressed on the endothelium, as well as measured the shear modulus and viscosity of the SS-RBC plasma membrane.

FRAME (Force Review Automation Environment): MATLAB-based AFM data processor.

Data processing of force-displacement curves generated by atomic force microscopes (AFMs) for elastic moduli and unbinding event measurements is very time consuming and susceptible to user error or bias. There is an evident need for consistent, dependable, and easy-to-use AFM data processing software. We have developed an open-source software application, the force review automation environment (or FRAME), that provides users with an intuitive graphical user interface, automating data processing, and tools for expediting manual processing. We did not observe a significant difference between manually processed and automatically processed results from the same data sets.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.