As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloprotei... more As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases.
CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobuli... more CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferati...
In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and ... more In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3-6 μm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a given pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be minimized by using ion selectivity of hydrogel-infiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity.
Inertial microfluidics has recently drawn wide attention as an efficient, high-throughput microfl... more Inertial microfluidics has recently drawn wide attention as an efficient, high-throughput microfluidic cell separation method. However, the achieved separation resolution and throughput, as well as the issues with cell dispersion due to cell-cell interaction, have appeared to be limiting factors in the application of the technique to real-world samples such as blood and other biological fluids. In this paper, we present a novel design of a spiral inertial microfluidic (trapezoidal cross-section) sorter with enhanced separation resolution and demonstrate its ability in separating/recovering polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (MNLs) from diluted human blood (1-2% hematocrit) with high efficiency (>80%). PMNs enriched by our method also showed negligible activation as compared to original input sample, while the conventional red blood cell (RBC) lysis method clearly induced artificial activation of the sensitive PMNs. Therefore, our proposed technique would be a promising alternative to enrich/separate sensitive blood cells for therapeutic or diagnostic applications.
As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloprotei... more As key components of autocrine signaling, pericellular proteases, a disintegrin and metalloproteinases (ADAMs) in particular, are known to impact the microenvironment of individual cells and have significant implications in various pathological situations including cancer, inflammatory and vascular diseases. There is great incentive to develop a high-throughput platform for single-cell measurement of pericellular protease activity, as it is essential for studying the heterogeneity of protease response and the corresponding cell behavioral consequences. In this work, we developed a microfluidic platform to simultaneously monitor protease activity of many single cells in a time-dependent manner. This platform isolates individual microwells rapidly on demand and thus allows single-cell activity measurement of both cell-surface and secreted proteases by confining individual cells with diffusive FRET-based substrates. With this platform, we observed dose-dependent heterogeneous protease activation of HepG2 cells treated with phorbol 12-myristate 13-acetate (PMA). To study the temporal behavior of PMA-induced protease response, we monitored the pericellular protease activity of the same single cells during three different time periods and revealed the diversity in the dynamic patterns of single-cell protease activity profile upon PMA stimulation. The unique temporal information of single-cell protease response can help unveil the complicated functional role of pericellular proteases.
CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobuli... more CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferati...
In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and ... more In this paper, we evaluate the strategy of using self-assembled microbeads to build a robust and tunable membrane for free-flow zone electrophoresis in a PDMS microfluidic chip. To fabricate a porous membrane as a salt bridge for free-flow zone electrophoresis, we used silica or polystyrene microbeads between 3-6 μm in diameter and packed them inside a microchannel. After complete evaporation, we infiltrated the porous microbead structure with a positively or negatively charged hydrogel to modify its surface charge polarity. Using this device, we demonstrated binary sorting (separation of positive and negative species at a given pH) of peptides and dyes in standard buffer systems without using sheath flows. The sample loss during sorting could be minimized by using ion selectivity of hydrogel-infiltrated microbead membranes. Our fabrication method enables building a robust membrane for pressure-driven free-flow zone electrophoresis with tunable pore size as well as surface charge polarity.
Inertial microfluidics has recently drawn wide attention as an efficient, high-throughput microfl... more Inertial microfluidics has recently drawn wide attention as an efficient, high-throughput microfluidic cell separation method. However, the achieved separation resolution and throughput, as well as the issues with cell dispersion due to cell-cell interaction, have appeared to be limiting factors in the application of the technique to real-world samples such as blood and other biological fluids. In this paper, we present a novel design of a spiral inertial microfluidic (trapezoidal cross-section) sorter with enhanced separation resolution and demonstrate its ability in separating/recovering polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (MNLs) from diluted human blood (1-2% hematocrit) with high efficiency (>80%). PMNs enriched by our method also showed negligible activation as compared to original input sample, while the conventional red blood cell (RBC) lysis method clearly induced artificial activation of the sensitive PMNs. Therefore, our proposed technique would be a promising alternative to enrich/separate sensitive blood cells for therapeutic or diagnostic applications.
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Papers by Lidan Wu