Single Cell Adhesion Assay Using Computer Controlled Micropipette

Abstract

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.

Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E‐selectin expression

“…Previously, it was applied to measure the adherence of macrophages and dendritic cells to fibrinogen, and to poly-L-Lys-graft-PEG. The adhesion force was in the same order of magnitude [35] as we found in the case of HUVECs/dPLB-985 cells. Our present data suggest that rMASP-1 induced high adhesion forces between dPLB-985 cells and HUVECs.…”

“…The possible role of E-selectin in this static model of dPLB-985 binding to HUVECs is further supported by the inhibition of adhesion by soluble E-selectin. To assess the adhesion force between rMASP-1-treated HUVECs and dPLB-985 cells, we utilized the recently introduced computer controlled micropipette method [34,35]. Previously, it was applied to measure the adherence of macrophages and dendritic cells to fibrinogen, and to poly-L-Lys-graft-PEG.…”

“…A computer controlled micropipette (CellSorter), mounted onto an inverted fluorescence microscope (Zeiss Axio Observer A1) [34,35], was used to quantify the adhesion force between the variously pre-treated HUVECs and dPLB-985 cells. In brief, a confluent layer of HUVECs was cultured in a 35-mm tissue culture plastic Petri dish (Greiner) for one day.…”

“…We used a computer controlled micropipette method to quantify the adhesion force between dPLB-985 cells and rMASP-1-treated HUVECs [34,35]. HUVECs were induced with 2 µM of rMASP-1, 300 nM thrombin for 6 hours, or 10 ng/mL TNFalpha for 24 hours or left untreated.…”

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

“…In the past decades, the field has attracted an increasingly broad scientific interest, and the interaction of either monocytes, MFs, or DCs with various proteins (including fibrinogen, 9,24 collagen-I, 25 laminin, 26 serum proteins, 27,28 and biomimetic peptides based on functional sites of fibronectin 29,30 ) have been investigated. However, lacking the appropriate technique, the overwhelming majority of previous studies failed to capture the inherent and essential dynamics of immune cell adhesion, and characterized it only at a single time point.…”